Pyrrolobenzodiazepine conjugates

ABSTRACT

A conjugate of formula (I), wherein L is a Ligand unit, D is a Drug Linker unit of formula (II), wherein either: (a) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (b) R10 is OH, and R11 is formula (A): p is an integer of from 1 to 20.

The present invention relates to conjugates comprising a specificpyrrolobenzodiazepine (PBD), and the precursor drug linker used to makesuch conjugates.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11 position which is the electrophilic centre responsiblefor alkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11(1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237(1986)). Their ability to form an adduct in the minor groove, enablesthem to interfere with DNA processing, hence their use as antitumouragents.

It has been previously disclosed that the biological activity of thismolecules can be potentiated by joining two PBD units together throughtheir C8/C′-hydroxyl functionalities via a flexible alkylene linker(Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992);Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBDdimers are thought to form sequence-selective DNA lesions such as thepalindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., etal., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al.,Biochemistry, 44, 4135-4147) which is thought to be mainly responsiblefor their biological activity.

One example of a PBD dimer is SG2000 (SJG-136):

(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C.,et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al.,Cancer Research, 64, 6693-6699 (2004)) which has been involved inclinical trials as a standalone agent, for example, NCT02034227investigating its use in treating Acute Myeloid Leukemia and ChronicLymphocytic Leukemia (see:https://www.clinicaltrials.gov/ct2/show/NCT02034227).

Dimeric PBD compounds bearing C2 aryl substituents, such as SG2202(ZC-207), are disclosed in WO 2005/085251:

and in WO2006/111759, bisulphites of such PBD compounds, for exampleSG2285 (ZC-423):

These compounds have been shown to be highly useful cytotoxic agents(Howard, P. W., et al., Bioorg. Med. Chem. (2009), doi:10.1016/j.bmcl.2009.09.012).

In an impact study submitted to the 2014 Research Excellence Framework(REF) in the United Kingdom by University College London (available athttp://impact.ref.ac.uk/casestudies2/refservice.svc/GetCaseStudyPDF/35393),it was commented that:

“The next generation of PBD dimers, which are more potent than SG2000,have been developed, including SG2057 and SG2202. They exhibitpicomolar/sub-picomolar activity against a range of human tumour celllines and demonstrate curative activity in human tumour xenograftmodels.” making reference to:

Hartley J A, et al., DNA interstrand cross-linking and in vivo antitumoractivity of the extended pyrrolo[2,1-c][1,4]benzodiazepine dimer SG2057.Invest New Drugs. 2012 June; 30(3):950-8.http://dx.doi.org/10.1007/s10637-011-9647-z (herein after “Hartley et al(2012)”) and:

“The ability to generate such cytotoxic molecules that display exquisitepotency suggested a potential role in strategies aimed at targeting andreleasing highly cytotoxic agents directly at a tumour site. An exampleis as the ‘warhead’ component of an antibody drug conjugate (ADC). Thefully synthetic PBD dimers are ideally suited for the role of warhead inan ADC approach.”

The Hartley et al (2012) paper comments in its summary that “SG2057 istherefore a highly active antitumour agent, with more potent in vitroactivity and superior in vivo activity to SG2000, warranting furtherdevelopment”.

SG2057 has the structure:

Antibody drug conjugates using SG2057 as a warhead were first disclosedin WO 2011/130598. For example, claim 54 of this application includesthe formula:

wherein n is from 1 to 24, more preferably 4 to 8. The following druglinkers were exemplified: n=4, 15c; n=8, 15d; n=24, 15e.

Claim 54 of this application also includes the formula:

wherein n is from 1 to 24, more preferably 4 to 8. The following druglinkers were exemplified: n=8, 58; n=24, 61.

WO 2011/130598 also discloses antibody-drug conjugates including thesedrug linkers, for example 110 (antiSteap1-15d), example 114(tastuzumab-15d) and example 115 (tastuzumab-58).

WO 2013/055987 discloses the drug linkers 14 and 22:

an their use in antibody-drug conjugates.

More recently, the warhead:

has been used in drug linkers and antibody-drug conjugates. WO2014/057074 discloses:

WO2015/052322 discloses:

DISCLOSURE OF THE INVENTION

The present inventors have surprisingly found that although SG2000 is atleast 10 times less cytotoxic than SG2057 (see Hartley et al 2012),particular antibody-drug conjugates appear to show at least comparableactivity. These conjugates have been shown to have surprisingly welltolerated in toxicity studies in a variety of species. This leads to theconjugates exhibiting high therapeutic indices and thus are promisingclinical candidates.

In a first aspect, the present invention provides Conjugates of formulaI:

L—(D^(L))_(p)  (I)

wherein L is a Ligand unit (i.e., a targeting agent), D is a Drug Linkerunit of formula II:

wherein

either:

(a) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogenand carbon atoms to which they are bound; or

(b) R¹⁰ is OH, and R¹¹ is:

p is an integer of from 1 to 20.

The Ligand unit, described more fully below, is a targeting agent thatbinds to a target moiety. The Ligand unit can, for example, specificallybind to a cell component (a Cell Binding Agent) or to other targetmolecules of interest. The Ligand unit can be, for example, a protein,polypeptide or peptide, such as an antibody, an antigen-binding fragmentof an antibody, or other binding agent, such as an Fc fusion protein.

A second aspect of the present invention provides a compound of formulaIII:

wherein

either:

(a) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogenand carbon atoms to which they are bound; or

(b) R¹⁰ is OH, and R¹¹ is:

A third aspect of the present invention provides the use of a conjugateof the first aspect of the invention in the manufacture of a medicamentfor treating a proliferative disease. The third aspect also provides aconjugate of the first aspect of the invention for use in the treatmentof a proliferative disease. The third aspect also provides a method oftreating a proliferative disease comprising administering atherapeutically effective amount of a conjugate of the first aspect ofthe invention to a patient in need thereof.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

A fourth aspect of the present invention provides the synthesis of aconjugate of the first aspect of the invention comprising conjugating acompound (drug linker) of the second aspect of the invention with aLigand Unit.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the effect on volume of a BT474 tumour following treatmentwith a conjugate of the present invention;

FIG. 2 shows the effect on volume of a BT474 tumour following treatmentwith a different conjugate of the present invention;

FIG. 3 shows the effect on volume of a NCI-N87 tumour followingtreatment with a conjugate of the present invention;

FIG. 4 shows the effect on volume of a NCI-N87 tumour followingtreatment with a different conjugate of the present invention.

D^(L)

In the first aspect D^(L) s selected from D^(L)-A and D^(L)-B:

In the second aspect, the compound is selected from A and B:

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide,peptide and a non-peptidic agent that specifically binds to a targetmolecule. In some embodiments, the Ligand unit may be a protein,polypeptide or peptide. In some embodiments, the Ligand unit may be acyclic polypeptide. These Ligand units can include antibodies or afragment of an antibody that contains at least one targetmolecule-binding site, lymphokines, hormones, growth factors, or anyother cell binding molecule or substance that can specifically bind to atarget.

The terms “specifically binds” and “specific binding” refer to thebinding of an antibody or other protein, polypeptide or peptide to apredetermined molecule (e.g., an antigen). Typically, the antibody orother molecule binds with an affinity of at least about 1×10⁻⁷ M⁻¹, andbinds to the predetermined molecule with an affinity that is at leasttwo-fold greater than its affinity for binding to a non-specificmolecule (e.g., BSA, casein) other than the predetermined molecule or aclosely-related molecule.

Examples of Ligand units include those agents described for use in WO2007/085930, which is incorporated herein.

In some embodiments, the Ligand unit is a Cell Binding Agent that bindsto an extracellular target on a cell. Such a Cell Binding Agent can be aprotein, polypeptide, peptide or a non-peptidic agent. In someembodiments, the Cell Binding Agent may be a protein, polypeptide orpeptide. In some embodiments, the Cell Binding Agent may be a cyclicpolypeptide. The Cell Binding Agent also may be antibody or anantigen-binding fragment of an antibody. Thus, in one embodiment, thepresent invention provides an antibody-drug conjugate (ADC).

Cell Binding Agent

A cell binding agent may be of any kind, and include peptides andnon-peptides. These can include antibodies or a fragment of an antibodythat contains at least one binding site, lymphokines, hormones, hormonemimetics, vitamins, growth factors, nutrient-transport molecules, or anyother cell binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptidecomprising 4-30, preferably 6-20, contiguous amino acid residues. Inthis embodiment, it is preferred that one cell binding agent is linkedto one monomer or dimer pyrrolobenzodiazepine compound.

In one embodiment the cell binding agent comprises a peptide that bindsintegrin α_(v)β₆. The peptide may be selective for α_(v)β₆ over XYS.

In one embodiment the cell binding agent comprises the A20FMDV-Cyspolypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC.Alternatively, a variant of the A20FMDV-Cys sequence may be used whereinone, two, three, four, five, six, seven, eight, nine or ten amino acidresidues are substituted with another amino acid residue. Furthermore,the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.

Antibodies

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin can be of anytype (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Theimmunoglobulins can be derived from any species, including human,murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and scFv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carryinga fully human immunoglobulin system (Lonberg (2008) Curr. Opinion20(4):450-459).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodiesinclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g. OldWorld Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g. human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and p, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Humanisation

Techniques to reduce the in vivo immunogenicity of a non-human antibodyor antibody fragment include those termed “humanisation”.

A “humanized antibody” refers to a polypeptide comprising at least aportion of a modified variable region of a human antibody wherein aportion of the variable region, preferably a portion substantially lessthan the intact human variable domain, has been substituted by thecorresponding sequence from a non-human species and wherein the modifiedvariable region is linked to at least another part of another protein,preferably the constant region of a human antibody. The expression“humanized antibodies” includes human antibodies in which one or morecomplementarity determining region (“CDR”) amino acid residues and/orone or more framework region (“FW” or “FR”) amino acid residues aresubstituted by amino acid residues from analogous sites in rodent orother non-human antibodies. The expression “humanized antibody” alsoincludes an immunoglobulin amino acid sequence variant or fragmentthereof that comprises an FR having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Or, looked at another way, a humanized antibody is ahuman antibody that also contains selected sequences from non-human(e.g. murine) antibodies in place of the human sequences. A humanizedantibody can include conservative amino acid substitutions ornon-natural residues from the same or different species that do notsignificantly alter its binding and/or biologic activity. Suchantibodies are chimeric antibodies that contain minimal sequence derivedfrom non-human immunoglobulins.

There are a range of humanisation techniques, including ‘CDR grafting’,‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known as‘veneering’), ‘composite antibodies’, ‘Human String ContentOptimisation’ and framework shuffling.

CDR Grafting

In this technique, the humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient antibody are replaced by residues from aCDR of a non-human species (donor antibody) such as mouse, rat, camel,bovine, goat, or rabbit having the desired properties (in effect, thenon-human CDRs are ‘grafted’ onto the human framework). In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues (this may happen when,for example, a particular FR residue has significant effect on antigenbinding).

Furthermore, humanized antibodies can comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. Thus, in general, a humanized antibody willcomprise all of at least one, and in one aspect two, variable domains,in which all or all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), or that of a human immunoglobulin.

Guided Selection

The method consists of combining the V_(H) or V_(L) domain of a givennon-human antibody specific for a particular epitope with a human V_(H)or V_(L) library and specific human V domains are selected against theantigen of interest. This selected human VH is then combined with a VLlibrary to generate a completely human VH×VL combination. The method isdescribed in Nature Biotechnology (N.Y.) 12, (1994) 899-903.

Composite Antibodies

In this method, two or more segments of amino acid sequence from a humanantibody are combined within the final antibody molecule. They areconstructed by combining multiple human VH and VL sequence segments incombinations which limit or avoid human T cell epitopes in the finalcomposite antibody V regions. Where required, T cell epitopes arelimited or avoided by, exchanging V region segments contributing to orencoding a T cell epitope with alternative segments which avoid T cellepitopes. This method is described in US 2008/0206239 A1.

Deimmunization

This method involves the removal of human (or other second species)T-cell epitopes from the V regions of the therapeutic antibody (or othermolecule). The therapeutic antibodies V-region sequence is analysed forthe presence of MHC class II-binding motifs by, for example, comparisonwith databases of MHC-binding motifs (such as the “motifs” databasehosted at www.wehi.edu.au). Alternatively, MHC class II-binding motifsmay be identified using computational threading methods such as thosedevised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in thesemethods, consecutive overlapping peptides from the V-region sequencesare testing for their binding energies to MHC class II proteins. Thisdata can then be combined with information on other sequence featureswhich relate to successfully presented peptides, such as amphipathicity,Rothbard motifs, and cleavage sites for cathepsin B and other processingenzymes.

Once potential second species (e.g. human) T-cell epitopes have beenidentified, they are eliminated by the alteration of one or more aminoacids. The modified amino acids are usually within the T-cell epitopeitself, but may also be adjacent to the epitope in terms of the primaryor secondary structure of the protein (and therefore, may not beadjacent in the primary structure). Most typically, the alteration is byway of substitution but, in some circumstances amino acid addition ordeletion will be more appropriate.

All alterations can be accomplished by recombinant DNA technology, sothat the final molecule may be prepared by expression from a recombinanthost using well established methods such as Site Directed Mutagenesis.However, the use of protein chemistry or any other means of molecularalteration is also possible.

Resurfacing

This method involves:

-   -   (a) determining the conformational structure of the variable        region of the non-human (e.g. rodent) antibody (or fragment        thereof) by constructing a three-dimensional model of the        non-human antibody variable region;    -   (b) generating sequence alignments using relative accessibility        distributions from x-ray crystallographic structures of a        sufficient number of non-human and human antibody variable        region heavy and light chains to give a set of heavy and light        chain framework positions wherein the alignment positions are        identical in 98% of the sufficient number of non-human antibody        heavy and light chains;    -   (c) defining for the non-human antibody to be humanized, a set        of heavy and light chain surface exposed amino acid residues        using the set of framework positions generated in step (b);    -   (d) identifying from human antibody amino acid sequences a set        of heavy and light chain surface exposed amino acid residues        that is most closely identical to the set of surface exposed        amino acid residues defined in step (c), wherein the heavy and        light chain from the human antibody are or are not naturally        paired;    -   (e) substituting, in the amino acid sequence of the non-human        antibody to be humanized, the set of heavy and light chain        surface exposed amino acid residues defined in step (c) with the        set of heavy and light chain surface exposed amino acid residues        identified in step (d);    -   (f) constructing a three-dimensional model of the variable        region of the non-human antibody resulting from the substituting        specified in step (e);    -   (g) identifying, by comparing the three-dimensional models        constructed in steps (a) and (f), any amino acid residues from        the sets identified in steps (c) or (d), that are within 5        Angstroms of any atom of any residue of the complementarity        determining regions of the non-human antibody to be humanized;        and    -   (h) changing any residues identified in step (g) from the human        to the original non-human amino acid residue to thereby define a        non-human antibody humanizing set of surface exposed amino acid        residues; with the proviso that step (a) need not be conducted        first, but must be conducted prior to step (g).

Superhumanization

The method compares the non-human sequence with the functional humangermline gene repertoire. Those human genes encoding canonicalstructures identical or closely related to the non-human sequences areselected. Those selected human genes with highest homology within theCDRs are chosen as FR donors. Finally, the non-human CDRs are graftedonto these human FRs. This method is described in patent WO 2005/079479A2.

Human String Content Optimization

This method compares the non-human (e.g. mouse) sequence with therepertoire of human germline genes and the differences are scored asHuman String Content (HSC) that quantifies a sequence at the level ofpotential MHC/T-cell epitopes. The target sequence is then humanized bymaximizing its HSC rather than using a global identity measure togenerate multiple diverse humanized variants (described in MolecularImmunology, 44, (2007) 1986-1998).

Framework Shuffling

The CDRs of the non-human antibody are fused in-frame to cDNA poolsencompassing all known heavy and light chain human germline geneframeworks. Humanised antibodies are then selected by e.g. panning ofthe phage displayed antibody library. This is described in Methods 36,43-60 (2005).

Examples of cell binding agents include those agents described for usein WO 2007/085930, which is incorporated herein.

Tumour-associate antigens and cognate antibodies for use in embodimentsof the present invention are listed below.

Tumor-Associated Antigens and Cognate Antibodies

(1) BMPR1B (Bone Morphogenetic Protein Receptor-Type IB)

Nucleotide

Genbank accession no. NM_001203

Genbank version no. NM_001203.2 GI:169790809

Genbank record update date: Sep. 23, 2012 02:06 PM

Polypeptide

Genbank accession no. NP_001194

Genbank version no. NP_001194.1 GI:4502431

Genbank record update date: Sep. 23, 2012 02:06 PM

CROSS-REFERENCES

ten Dijke, P., et al Science 264 (5155): 101-104 (1994), Oncogene 14(11):1377-1382 (1997)); WO2004/063362 (Claim 2); WO2003/042661 (Claim12); US2003/134790-A1 (Page 38-39); WO2002/102235 (Claim 13; Page 296);WO2003/055443 (Page 91-92); WO2002/99122 (Example 2; Page 528-530);WO2003/029421 (Claim 6); WO2003/024392 (Claim 2; FIG. 112); WO2002/98358(Claim 1; Page 183); WO2002/54940 (Page 100-101); WO2002/59377 (Page349-350); WO2002/30268 (Claim 27; Page 376); 15 WO2001/48204 (Example;FIG. 4); NP_001194 bone morphogenetic protein receptor, typeIB/pid=NP_001194.1; MIM:603248; AY065994

(2) E16 (LAT1, SLC7A5)

Nucleotide

Genbank accession no. NM_003486

Genbank version no. NM_003486.5 GI:71979931

Genbank record update date: Jun. 27, 2012 12:06 PM

Polypeptide

Genbank accession no. NP_003477

Genbank version no. NP_003477.4 GI:71979932

Genbank record update date: Jun. 27, 2012 12:06 PM

CROSS REFERENCES

-   Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395    (6699):288-291 (1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem.    267 (16):11267-11273); WO2004/048938 (Example 2); WO2004/032842    (Example IV); WO2003/042661 (Claim 12); WO2003/016475 (Claim 1);    WO2002/78524 (Example 2); WO2002/99074 (Claim 19; Page 127-129);    WO2002/86443 (Claim 27; Pages 222, 393); WO2003/003906 (Claim 10;    Page 293); WO2002/64798 (Claim 33; Page 93-95); WO2000/14228 (Claim    5; Page 133-136); US2003/224454 (FIG. 3); WO2003/025138 (Claim 12;    Page 150); NP_003477 solute carrier family 7 (cationic amino acid    transporter, y+system), member 5/pid=NP_003477.3—Homo sapiens;    MIM:600182; NM_015923.

(3) STEAP1 (Six Transmembrane Epithelial Antigen of Prostate)

Nucleotide

Genbank accession no. NM_012449

Genbank version no. NM_012449.2 GI:22027487

Genbank record update date: Sep. 9, 2012 02:57 PM

Polypeptide

Genbank accession no. NP_036581

Genbank version no. NP_036581.1 GI:9558759

Genbank record update date: Sep. 9, 2012 02:57 PM

CROSS REFERENCES

-   Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999)    Proc. Nat. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004/065577    (Claim 6); WO2004/027049 (FIG. 1L); EP1394274 (Example 11);    WO2004/016225 (Claim 2); WO2003/042661 (Claim 12); US2003/157089    (Example 5); US2003/185830 (Example 5); US2003/064397 (FIG. 2);    WO2002/89747 (Example 5; Page 618-619); WO2003/022995 (Example 9;    FIG. 13A, Example 53; Page 173, Example 2; FIG. 2A); six    transmembrane epithelial antigen of the prostate; MIM:604415.

(4) 0772P (CA125, MUC16)

Nucleotide

Genbank accession no. AF361486

Genbank version no. AF361486.3 GI:34501466

Genbank record update date: Mar. 11, 2010 07:56 AM

Polypeptide

Genbank accession no. AAK74120

Genbank version no. AAK74120.3 GI:34501467

Genbank record update date: Mar. 11, 2010 07:56 AM

CROSS REFERENCES

-   J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004/045553 (Claim    14); WO2002/92836 (Claim 6; FIG. 12); WO2002/83866 (Claim 15; Page    116-121); US2003/124140 (Example 16); GI:34501467;

(5) MPF (MPF, MSLN, SMR, Megakaryocyte Potentiating Factor, Mesothelin)

Nucleotide

Genbank accession no. NM_005823

Genbank version no. NM_005823.5 GI:293651528

Genbank record update date: Sep. 2, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_005814

Genbank version no. NP_005814.2 GI:53988378

Genbank record update date: Sep. 2, 2012 01:47 PM

CROSS REFERENCES

-   Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc.    Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999), Proc. Natl.    Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol. Chem. 270    (37):21984-21990 (1995)); WO2003/101283 (Claim 14); (WO2002/102235    (Claim 13; Page 287-288); WO2002/101075 (Claim 4; Page 308-309);    WO2002/71928 (Page 320-321); WO94/10312 (Page 52-57); IM:601051.

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, Solute Carrier Family 34 (SodiumPhosphate), Member 2, Type II Sodium-Dependent Phosphate Transporter 3b)

Nucleotide

Genbank accession no. NM_006424

Genbank version no. NM_006424.2 GI:110611905

Genbank record update date: Jul. 22, 2012 03:39 PM

Polypeptide

Genbank accession no. NP_006415

Genbank version no. NP_006415.2 GI:110611906

Genbank record update date: Jul. 22, 2012 03:39 PM

CROSS REFERENCES

J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284(1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004/022778 (Claim 2); EP1394274 (Example 11);WO2002/102235 (Claim 13; Page 326); EP0875569 (Claim 1; Page 17-19);WO2001/57188 (Claim 20; Page 329); WO2004/032842 (Example IV);WO2001/75177 (Claim 24; Page 139-140); MIM:604217.

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, 25 Sema Domain, Seven Thrombospondin Repeats (Type 1 and Type1-Like), Transmembrane Domain™ and Short Cytoplasmic Domain,(Semaphorin) 5B)

Nucleotide

Genbank accession no. AB040878

Genbank version no. AB040878.1 GI:7959148

Genbank record update date: Aug. 2, 2006 05:40 PM

Polypeptide

Genbank accession no. BAA95969

Genbank version no. BAA95969.1 GI:7959149

Genbank record update date: Aug. 2, 2006 05:40 PM

CROSS REFERENCES

Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004/000997 (Claim1); WO2003/003984 (Claim 1); WO2002/06339 (Claim 1; Page 50);WO2001/88133 (Claim 1; Page 41-43, 48-58); WO2003/054152 (Claim 20);WO2003/101400 (Claim 11); Accession: 30 Q9P283; Genew; HGNC:10737

(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 Gene)

Nucleotide

Genbank accession no. AY358628

Genbank version no. AY358628.1 GI:37182377

Genbank record update date: Dec. 1, 2009 04:15 AM

Polypeptide

Genbank accession no. AAQ88991

Genbank version no. AAQ88991.1 GI:37182378

Genbank record update date: Dec. 1, 2009 04:15 AM

CROSS REFERENCES

Ross et al (2002) Cancer Res. 62:2546-2553; US2003/129192 (Claim 2);US2004/044180 (Claim 12); US2004/044179 (Claim 11); US2003/096961 (Claim11); US2003/232056 (Example 5); WO2003/105758 16 (Claim 12);US2003/206918 (Example 5); EP1347046 (Claim 1); WO2003/025148 (Claim20); GI:37182378.

(9) ETBR (Endothelin Type B Receptor)

Nucleotide

Genbank accession no. AY275463

Genbank version no. AY275463.1 GI:30526094

Genbank record update date: Mar. 11, 2010 02:26 AM

Polypeptide

Genbank accession no. AAP32295

Genbank version no. AAP32295.1 GI:30526095

Genbank record update date: Mar. 11, 2010 02:26 AM

CROSS REFERENCES

Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255,1991; AraiH., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et alEur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Ce/79,1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et alHum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet.12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002)Hum. Genet. 111, 198-206; WO2004/045516 (Claim 1); WO2004/048938(Example 2); WO2004/040000 (Claim 151); WO2003/087768 (Claim 1); 20WO2003/016475 (Claim 1); WO2003/016475 (Claim 1); WO2002/61087 (FIG. 1);WO2003/016494 (FIG. 6); WO2003/025138 (Claim 12; Page 144); WO2001/98351(Claim 1; Page 124-125); EP0522868 (Claim 8; FIG. 2); WO2001/77172(Claim 1; Page 297-299); US2003/109676; U.S. Pat. No. 6,518,404 (FIG.3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34); WO2004/001004.

(10) MSG783 (RNF124, hypothetical protein FLJ20315)

Nucleotide

Genbank accession no. NM_017763

Genbank version no. NM_017763.4 GI:167830482

Genbank record update date: Jul. 22, 2012 12:34 AM

Polypeptide

Genbank accession no. NP_060233

Genbank version no. NP_060233.3 GI:56711322

Genbank record update date: Jul. 22, 2012 12:34 AM

CROSS REFERENCES

WO2003/104275 (Claim 1); WO2004/046342 (Example 2); WO2003/042661 (Claim12); WO2003/083074 (Claim 14; Page 61); WO2003/018621 (Claim 1);WO2003/024392 (Claim 2; FIG. 93); WO2001/66689 (Example 6);LocusD:54894.

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, ProstateCancer Associated Gene 1, Prostate Cancer Associated Protein 1, SixTransmembrane Epithelial Antigen of Prostate 2, Six TransmembraneProstate Protein)

Nucleotide

Genbank accession no. AF455138

Genbank version no. AF455138.1 GI:22655487

Genbank record update date: Mar. 11, 2010 01:54 AM

Polypeptide

Genbank accession no. AAN04080

Genbank version no. AAN04080.1 GI:22655488

Genbank record update date: Mar. 11, 2010 01:54 AM

CROSS REFERENCES

Lab. Invest. 82 (11):1573-1582 (2002)); WO2003/087306; US2003/064397(Claim 1; FIG. 1); WO2002/72596 (Claim 13; Page 54-55); WO2001/72962(Claim 1; FIG. 4B); WO2003/104270 (Claim 11); WO2003/104270 (Claim 16);US2004/005598 (Claim 22); WO2003/042661 (Claim 12); US2003/060612 (Claim12; FIG. 10); WO2002/26822 (Claim 23; FIG. 2); WO2002/16429 (Claim 12;FIG. 10); GI:22655488.

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, Transient ReceptorPotential Cation 5 Channel, Subfamily M, Member 4)

Nucleotide

Genbank accession no. NM_017636

Genbank version no. NM_017636.3 GI:304766649

Genbank record update date: Jun. 29, 2012 11:27 AM

Polypeptide

Genbank accession no. NP_060106

Genbank version no. NP_060106.2 GI:21314671

Genbank record update date: Jun. 29, 2012 11:27 AM

CROSS REFERENCES

Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697(2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820(2003)); US2003/143557 (Claim 4); WO2000/40614 (Claim 14; Page 100-103);WO2002/10382 (Claim 1; FIG. 9A); WO2003/042661 (Claim 12); WO2002/30268(Claim 27; Page 391); US2003/219806 (Claim 4); WO2001/62794 (Claim 14;FIG. 1A-D); MIM:606936.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor)

Nucleotide

Genbank accession no. NM_003212

Genbank version no. NM_003212.3 GI:292494881

Genbank record update date: Sep. 23, 2012 02:27 PM

Polypeptide

Genbank accession no. NP_003203

Genbank version no. NP_003203.1 GI:4507425

Genbank record update date: Sep. 23, 2012 02:27 PM

CROSS REFERENCES

Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003/224411 (Claim 1); WO2003/083041(Example 1); WO2003/034984 (Claim 12); WO2002/88170 (Claim 2; Page52-53); WO2003/024392 (Claim 2; FIG. 58); WO2002/16413 (Claim 1; Page94-95,105); WO2002/22808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399(Example 2; Col 17-18); U.S. Pat. No. 5,792,616 (FIG. 2); MIM:187395.

(14) CD21 (CR2 (Complement Receptor 2) or C3DR (C3d/Epstein Barr VirusReceptor) or Hs.73792)

Nucleotide

Genbank accession no M26004

Genbank version no. M26004.1 GI:181939

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35786

Genbank version no. AAA35786.1 GI:181940

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., etal J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Nat. Acad.Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al Proc. Nat. Acad. Sci. U.S.A. 83,5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320;WO2004/045520 (Example 4); US2004/005538 (Example 1); WO2003/062401(Claim 9); WO2004/045520 (Example 4); WO91/02536 (FIGS. 9.1-9.9);WO2004/020595 (Claim 1); Accession: P20023; Q13866; Q14212; EMBL;M26004; AAA35786.1.

(15) CD79b (CD79B, CD79p, IGb (Immunoglobulin-Associated Beta), B29)

Nucleotide

Genbank accession no NM_000626

Genbank version no. NM_000626.2 GI:90193589

Genbank record update date: Jun. 26, 2012 01:53 PM

Polypeptide

Genbank accession no. NP_000617

Genbank version no. NP 000617.1 GI:11038674

Genbank record update date: Jun. 26, 2012 01:53 PM

CROSS REFERENCES

Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625);WO2004/016225 (claim 2, FIG. 140); WO2003/087768, US2004/101874 (claim1, page 102); WO2003/062401 (claim 9); WO2002/78524 (Example 2);US2002/150573 (claim 35 5, page 15); U.S. Pat. No. 5,644,033;WO2003/048202 (claim 1, pages 306 and 309); WO 99/58658, U.S. Pat. No.6,534,482 (claim 13, FIG. 17A/B); WO2000/55351 (claim 11, pages1145-1146); MIM:147245

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 Domain Containing PhosphataseAnchor Protein 5 1a), SPAP1B, SPAP1C)

Nucleotide

Genbank accession no NM_030764

Genbank version no. NM_030764.3 GI:227430280

Genbank record update date: Jun. 30, 2012 12:30 AM

Polypeptide

Genbank accession no. NP_110391

Genbank version no. NP_110391.2 GI:19923629

Genbank record update date: Jun. 30, 2012 12:30 AM

CROSS REFERENCES

AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci.U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem.Biophys. Res. Commun. 280 (3):768-775; WO2004/016225 (Claim 2);WO2003/077836; WO2001/38490 (Claim 5; FIG. 18D-1-18D-2); WO2003/097803(Claim 12);

10 WO2003/089624 (Claim 25): MIM:606509.

(17) HER2 (ErbB2)

Nucleotide

Genbank accession no M11730

Genbank version no. M11730.1 GI:183986

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA75493

Genbank version no. AAA75493.1 GI:306840

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T., etal Nature 319, 230-234, 1986; Semba K., et al Proc. Nat. Acad. Sci.U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165,869-880, 2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999;Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993)Genomics 15, 426-429; WO2004/048938 (Example 2); WO2004/027049 (FIG.11); WO2004/009622; WO2003/081210; WO2003/089904 (Claim 9);WO2003/016475 (Claim 1); US2003/118592; WO2003/008537 (Claim 1);WO2003/055439 (Claim 29; FIG. 1A-B); WO2003/025228 (Claim 37; FIG. 5C);20 WO2002/22636 (Example 13; Page 95-107); WO2002/12341 (Claim 68; FIG.7); WO2002/13847 (Page 71-74); WO2002/14503 (Page 114-117); WO2001/53463(Claim 2; Page 41-46); WO2001/41787 (Page 15); WO2000/44899 (Claim 52;FIG. 7); WO2000/20579 (Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim3; Col 31-38); WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7);WO2004/043361 (Claim 7); WO2004/022709; WO2001/00244 25 (Example 3; FIG.4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761;AAA35808.1

Antibodies

Abbott: US20110177095

-   -   For example, an antibody comprising CDRs having overall at least        80% sequence identity to CDRs having amino acid sequences of SEQ        ID NO:3 (CDR-H1), SEQ ID NO:4 (CDR-H2), SEQ ID NO:5 (CDR-H3),        SEQ ID NO:104 and/or SEQ ID NO:6 (CDR-L1), SEQ ID NO:7 (CDR-L2),        and SEQ ID NO:8 (CDR-L3), wherein the anti-HER2 antibody or        anti-HER2 binding fragment has reduced immunogenicity as        compared to an antibody having a VH of SEQ ID NO:1 and a VL of        SEQ ID NO:2.

Biogen: US20100119511

-   -   For example, ATCC accession numbers: PTA-10355, PTA-10356,        PTA-10357, PTA-10358    -   For example, a purified antibody molecule that binds to HER2        comprising a all six CDR's from an antibody selected from the        group consisting of BIIB71F10 (SEQ ID NOs:11, 13), BIIB69A09        (SEQ ID NOs:15, 17); BIIB67F10 (SEQ ID NOs:19, 21); BIIB67F11        (SEQ ID NOs:23, 25), BIIB66A12 (SEQ ID NOs:27, 29), BIIB66C01        (SEQ ID NOs:31, 33), BIIB65C10 (SEQ ID NOs:35, 37), BIIB65H09        (SEQ ID NOs:39, 41) and BIIB65B03 (SEQ ID NOs:43, 45), or CDRs        which are identical or which have no more than two alterations        from said CDRs.

Herceptin (Genentech)—U.S. Pat. No. 6,054,297; ATCC accession no.CRL-10463 (Genentech)

Pertuzumab (Genentech)

-   -   US20110117097        -   for example, see SEQ IDs No. 15&16, SEQ IDs No. 17&18, SEQ            IDs No. 23&24 & ATCC accession numbers HB-12215, HB-12216,            CRL 10463, HB-12697.    -   US20090285837    -   US20090202546        -   for example, ATCC accession numbers: HB-12215, HB-12216, CRL            10463, H B-12698.    -   US20060088523        -   for example, ATCC accession numbers: HB-12215, HB-12216        -   for example, an antibody comprising the variable light and            variable heavy amino acid sequences in SEQ ID Nos. 3 and 4,            respectively.        -   for example, an antibody comprising a light chain amino acid            sequence selected from SEQ ID No. 15 and 23, and a heavy            chain amino acid sequence selected from SEQ ID No. 16 and 24    -   US20060018899        -   for example, ATCC accession numbers: (7C2) HB-12215, (7F3)            HB-12216, (4D5) CRL-10463, (2C4) HB-12697.        -   for example, an antibody comprising the amino acid sequence            in SEQ ID No. 23, or a deamidated and/or oxidized variant            thereof.    -   US2011/0159014        -   for example, an antibody having a light chain variable            domain comprising the hypervariable regions of SEQ ID NO:            1”.        -   For example, an antibody having a heavy chain variable            domain comprising the hypervariable regions of SEQ ID NO: 2.    -   US20090187007

Glycotope: TrasGEX antibody http://www.glycotope.com/pipeline

-   -   For example, see International Joint Cancer Institute and        Changhai Hospital Cancer Cent: HMTI-Fc Ab—Gao J., et al BMB Rep.        2009 Oct. 31; 42(10):636-41.

Symphogen: US20110217305

Union Stem Cell &Gene Engineering, China—Liu H Q., et al XiBao YuFenZiMian YiXue Za Zhi. 2010 May; 26(5):456-8.

(18) NCA (CEACAM6)

Nucleotide

Genbank accession no M18728

Genbank version no. M18728.1 GI:189084

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59907

Genbank version no. AAA59907.1 GI:189085

Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem.Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc.Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004/063709; EP1439393(Claim 7); WO2004/044178 (Example 4); WO2004/031238; WO2003/042661(Claim 12); WO2002/78524 (Example 2); WO2002/86443 (Claim 27; Page 427);WO2002/60317 (Claim 2); Accession: P40199; Q14920; EMBL; M29541;AAA59915.1.

EMBL; M18728.

(19) MDP (DPEP1)

Nucleotide

Genbank accession no BC017023

Genbank version no. BC017023.1 GI:16877538

Genbank record update date: Mar. 6, 2012 01:00 PM

Polypeptide

Genbank accession no. AAH17023

Genbank version no. AAH17023.1 GI:16877539

Genbank record update date: Mar. 6, 2012 01:00 PM

CROSS REFERENCES

Proc. Nat. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003/016475(Claim 1); WO2002/64798 (Claim 33; Page 85-87); JP05003790 (FIG. 6-8);WO99/46284 (FIG. 9); MIM:179780.

(20) IL20R-alpha (IL20Ra, ZCYTOR7)

Nucleotide

Genbank accession no AF184971

Genbank version no. AF184971.1 GI:6013324

Genbank record update date: Mar. 10, 2010 10:00 PM

Polypeptide

Genbank accession no. AAF01320

Genbank version no. AAF01320.1 GI:6013325

Genbank record update date: Mar. 10, 2010 10:00 PM

CROSS REFERENCES

Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et alNature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001;Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J.,et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003) 10Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004/005320 (Example 5);WO2003/029262 (Page 74-75); WO2003/002717 (Claim 2; Page 63);WO2002/22153 (Page 45-47); US2002/042366 (Page 20-21); WO2001/46261(Page 57-59); WO2001/46232 (Page 63-65); WO98/37193 (Claim 1; Page55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB)

Nucleotide

Genbank accession no AF229053

Genbank version no. AF229053.1 GI:10798902

Genbank record update date: Mar. 11, 2010 12:58 AM

Polypeptide

Genbank accession no. AAG23135

Genbank version no. AAG23135.1 GI:10798903

Genbank record update date: Mar. 11, 2010 12:58 AM

CROSS REFERENCES

Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al GenomeRes. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Nat. Acad. Sci.U.S.A. 99, 16899-16903, 2002; US2003/186372 (Claim 11); US2003/186373(Claim 11); US2003/119131 (Claim 1; FIG. 52); US2003/119122 (Claim 1;FIG. 52); US2003/119126 (Claim 1); US2003/119121 (Claim 1; FIG. 52);US2003/119129 (Claim 1); US2003/119130 (Claim 1); US2003/119128 (Claim1; FIG. 52); US2003/119125 (Claim 1); WO2003/016475 (Claim 1);WO2002/02634 (Claim 1)

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5)

Nucleotide

Genbank accession no NM_004442

Genbank version no. NM_004442.6 GI:111118979

Genbank record update date: Sep. 8, 2012 04:43 PM

Polypeptide

Genbank accession no. NP_004433

Genbank version no. NP 004433.2 GI:21396504

Genbank record update date: Sep. 8, 2012 04:43 PM

CROSS REFERENCES

Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10(5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev.Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12); WO200053216 (Claim1; Page 41); WO2004065576 (Claim 1); WO2004020583 (Claim 9);WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42); MIM:600997.

(23) ASLG659 (B7h)

Nucleotide

Genbank accession no. AX092328

Genbank version no. AX092328.1 GI:13444478

Genbank record update date: Jan. 26, 2011 07:37 AM

CROSS REFERENCES

US2004/0101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (FIG.3); US2003/165504 (Claim 1); US2003/124140 (Example 2); US2003/065143(FIG. 60); WO2002/102235 (Claim 13; Page 299); US2003/091580 (Example2); WO2002/10187 (Claim 6; FIG. 10); WO2001/94641 (Claim 12; FIG. 7b);WO2002/02624 (Claim 13; FIG. 1A-1B); US2002/034749 (Claim 54; Page45-46); WO2002/06317 (Example 2; Page 320-321, Claim 34; Page 321-322);WO2002/71928 (Page 468-469); WO2002/02587 (Example 1; FIG. 1);WO2001/40269 (Example 3; Pages 190-192); WO2000/36107 (Example 2; Page205-207); WO2004/053079 (Claim 12); WO2003/004989 (Claim 1);WO2002/71928 (Page 233-234, 452-453); WO 01/16318.

(24) PSCA (Prostate stem cell antigen precursor)

Nucleotide

Genbank accession no AJ297436

Genbank version no. AJ297436.1 GI:9367211

Genbank record update date: Feb. 1, 2011 11:25 AM

Polypeptide

Genbank accession no. CAB97347

Genbank version no. CAB97347.1 GI:9367212

Genbank record update date: Feb. 1, 2011 11:25 AM

CROSS REFERENCES

Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998;Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res.Commun. (2000) 275(3):783-788; WO2004/022709; EP1394274 (Example 11);US2004/018553 (Claim 17); WO2003/008537 (Claim 1); WO2002/81646 (Claim1; Page 164); WO2003/003906 (Claim 10; Page 288); WO2001/40309 (Example1; FIG. 17); US2001/055751 (Example 1; FIG. 1b ); WO2000/32752 (Claim18; FIG. 1); WO98/51805 (Claim 17; Page 97); WO98/51824 (Claim 10; Page94); WO98/40403 (Claim 2; FIG. 1B); Accession: 043653; EMBL; AF043498;AAC39607.1

(25) GEDA

Nucleotide

Genbank accession no AY260763

Genbank version no. AY260763.1 GI:30102448

Genbank record update date: Mar. 11, 2010 02:24 AM

Polypeptide

Genbank accession no. AAP14954

Genbank version no. AAP14954.1 GI:30102449

Genbank record update date: Mar. 11, 2010 02:24 AM

CROSS REFERENCES

AP14954 lipoma HMGIC fusion-partnerlike protein/pid=AAP14954.1—Homosapiens (human); WO2003/054152 (Claim 20); WO2003/000842 (Claim 1);WO2003/023013 (Example 3, Claim 20); US2003/194704 (Claim 45);GI:30102449;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3)

Nucleotide

Genbank accession no AF116456

Genbank version no. AF116456.1 GI:4585274

Genbank record update date: Mar. 10, 2010 09:44 PM

Polypeptide

Genbank accession no. AAD25356

Genbank version no. AAD25356.1 GI:4585275

Genbank record update date: Mar. 10, 2010 09:44 PM

CROSS REFERENCES

BAFF receptor/pid=NP_443177.1—Homo sapiens: Thompson, J. S., et alScience 293 (5537), 2108-2111 (2001); WO2004/058309; WO2004/011611;WO2003/045422 (Example; Page 32-33); WO2003/014294 (Claim 35; FIG. 6B);WO2003/035846 (Claim 70; Page 615-616); WO2002/94852 (Col 136-137);WO2002/38766 (Claim 3; Page 133); WO2002/24909 (Example 3; FIG. 3);MIM:606269; NP_443177.1; NM_052945_1; AF132600

(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814)

Nucleotide

Genbank accession no AK026467

Genbank version no. AK026467.1 GI:10439337

Genbank record update date: Sep. 11, 2006 11:24 PM

Polypeptide

Genbank accession no. BAB15489

Genbank version no. BAB15489.1 GI:10439338

Genbank record update date: Sep. 11, 2006 11:24 PM

CROSS REFERENCES

Wilson et al (1991) J. Exp. Med. 173:137-146; WO2003/072036 (Claim 1;FIG. 1); IM:107266; NP_001762.1; NM_001771_1.

(27a) CD22 (CD22 molecule)

Nucleotide

Genbank accession no X52785

Genbank version no. X52785.1 GI:29778

Genbank record update date: Feb. 2, 2011 10:09 AM

Polypeptide

Genbank accession no. CAA36988

Genbank version no. CAA36988.1 GI:29779

Genbank record update date: Feb. 2, 2011 10:09 AM

CROSS REFERENCES

Stamenkovic I. et al., Nature 345 (6270), 74-77 (1990)??

Other Information

Official Symbol: CD22

Other Aliases: SIGLEC-2, SIGLEC2

Other Designations: B-cell receptor CD22; B-lymphocyte cell adhesionmolecule; BL-CAM; CD22 antigen; T-cell surface antigen Leu-14; sialicacid binding Ig-like lectin 2; sialic acid-binding Ig-like lectin 2

Antibodies

G5/44 (Inotuzumab): DiJoseph J F., et al Cancer Immunol Immunother. 2005January; 54(1):11-24.

Epratuzumab—Goldenberg D M., et al Expert Rev Anticancer Ther. 6(10):1341-53, 2006.

(28) CD79a (CD79A, CD79alpha), Immunoglobulin-Associated Alpha, a BCell-Specific Protein that Covalently Interacts with Ig Beta (CD79B) andForms a Complex on the Surface with Ig M 35 molecules, transduces asignal involved in B-cell differentiation), pI: 4.84, MW: 25028 TM: 2

[P] Gene Chromosome: 19q13.2).

Nucleotide

Genbank accession no NM_001783

Genbank version no. NM_001783.3 GI:90193587

Genbank record update date: Jun. 26, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001774

Genbank version no. NP_001774.1 GI:4502685

Genbank record update date: Jun. 26, 2012 01:48 PM

CROSS REFERENCES

WO2003/088808, US2003/0228319; WO2003/062401 (claim 9); US2002/150573(claim 4, pages 13-14); WO99/58658 (claim 13, FIG. 16); WO92/07574 (FIG.1); U.S. Pat. No. 5,644,033; Ha et al (1992) J. Immunol.148(5):1526-1531; Müller et al (1992) Eur. J. Immunol. 22:1621-1625;Hashimoto et al (1994) Immunogenetics 40(4):287-295; Preud'homme et al(1992) Clin. Exp. 5 Immunol. 90(1):141-146; Yu et al (1992) J. Immunol.148(2) 633-637; Sakaguchi et al (1988) EMBO J. 7(11):3457-3464

(29) CXCR5 (Burkitt's Lymphoma Receptor 1, a G Protein-Coupled Receptorthat is Activated by the CXCL13 Chemokine, Functions in LymphocyteMigration and Humoral Defense, Plays a 10 Role in HIV-2 Infection andPerhaps Development of AIDS, Lymphoma, Myeloma, and Leukemia); 372 aa,pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3,

Nucleotide

Genbank accession no NM_001716

Genbank version no. NM_001716.4 GI:342307092

Genbank record update date: Sep. 30, 2012 01:49 PM

Polypeptide

Genbank accession no. NP_001707

Genbank version no. NP_001707.1 GI:4502415

Genbank record update date: Sep. 30, 2012 01:49 PM

CROSS REFERENCES

WO2004/040000; WO2004/015426; US2003/105292 (Example 2); U.S. Pat. No.6,555,339 (Example 2); WO2002/61087 (FIG. 1); WO2001/57188 (Claim 20,page 269); WO2001/72830 (pages 12-13); WO2000/22129 (Example 1, pages152-153, Example 2, pages 254-256); WO99/28468 (claim 1, page 38); U.S.Pat. No. 5,440,021 (Example 2, col 49-52); WO94/28931 (pages 56-58);WO92/17497 (claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol.22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779

(30) HLA-DOB (Beta Subunit of MHC Class II Molecule (La Antigen) thatBinds Peptides and 20 Presents them to CD4+ T Lymphocytes); 273 aa, pI:6.56, MW: 30820.TM: 1 [P] Gene Chromosome: 6p21.3)

Nucleotide

Genbank accession no NM_002120

Genbank version no. NM_002120.3 GI:118402587

Genbank record update date: Sep. 8, 2012 04:46 PM

Polypeptide

Genbank accession no. NP_002111

Genbank version no. NP_002111.1 GI:4504403

Genbank record update date: Sep. 8, 2012 04:46 PM

CROSS REFERENCES

Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989)Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) TissueAntigens 59:512-519; WO99/58658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).

Nucleotide

Genbank accession no NM_002561

Genbank version no. NM_002561.3 GI:325197202

Genbank record update date: Jun. 27, 2012 12:41 AM

Polypeptide

Genbank accession no. NP_002552

Genbank version no. NP_002552.2 GI:28416933

Genbank record update date: Jun. 27, 2012 12:41 AM

CROSS REFERENCES

Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004/047749;WO2003/072035 (claim 10); Touchman et al (2000) Genome Res. 10:165-173;WO2002/22660 (claim 20); WO2003/093444 (claim 1); WO2003/087768 (claim1); WO2003/029277 (page 82) (32) CD72 (B-cell differentiation antigenCD72, Lyb-2); 359 aa, pI: 8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome:9p13.3).

Nucleotide

Genbank accession no NM_001782

Genbank version no. NM_001782.2 GI:194018444

Genbank record update date: Jun. 26, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_001773

Genbank version no. NP_001773.1 GI:4502683

Genbank record update date: Jun. 26, 2012 01:43 PM

CROSS REFERENCES

WO2004042346 (claim 65); WO2003/026493 (pages 51-52, 57-58);WO2000/75655 (pages 105-106); Von Hoegen et al (1990) J. Immunol.144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA99:16899-16903.

(33) LY64 (Lymphocyte Antigen 64 (RP105), Type I Membrane Protein of theLeucine Rich Repeat (LRR) Family, Regulates B-Cell Activation andApoptosis, Loss of Function is Associated with Increased DiseaseActivity in Patients with Systemic Lupus Erythematosis); 661 Aa, pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).

Nucleotide

Genbank accession no NM_005582

Genbank version no. NM_005582.2 GI:167555126

Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_005573

Genbank version no. NP_005573.2 GI:167555127

Genbank record update date: Sep. 2, 2012 01:50 PM

CROSS REFERENCES

US2002/193567; WO97/07198 (claim 11, pages 39-42); Miura et al (1996)Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822;WO2003/083047; WO97/44452 (claim 8, pages 57-61); WO2000/12130 (pages24-26).

(34) FcRH1 (Fc Receptor-Like Protein 1, a Putative Receptor for theImmunoglobulin Fc Domain that Contains C2 Type Ig-Like and ITAM Domains,May have a Role in B-Lymphocyte 20 Differentiation); 429 aa, pI: 5.28,MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22)

Nucleotide

Genbank accession no NM_052938

Genbank version no. NM_052938.4 GI:226958543

Genbank record update date: Sep. 2, 2012 01:43 PM

Polypeptide

Genbank accession no. NP_443170

Genbank version no. NP_443170.1 GI:16418419

Genbank record update date: Sep. 2, 2012 01:43 PM

CROSS REFERENCES

WO2003/077836; WO2001/38490 (claim 6, FIG. 18E-1-18-E-2); Davis et al(2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003/089624 (claim8); EP1347046 (claim 1); WO2003/089624 (claim 7).

(35) IRTA2 (Immunoglobulin Superfamily Receptor Translocation Associated2, a Putative Immunoreceptor with Possible Roles in B Cell Developmentand Lymphomagenesis; Deregulation of the Gene by Translocation Occurs inSome B Cell Malignancies); 977 Aa, pI: 6.88, MW: 106468, TM: 1 [P] GeneChromosome: 1q21)

Nucleotide

Genbank accession no AF343662

Genbank version no. AF343662.1 G:13591709

Genbank record update date: Mar. 11, 2010 01:16 AM

Polypeptide

Genbank accession no. AAK31325

Genbank version no. AAK31325.1 GI:13591710

Genbank record update date: Mar. 11, 2010 01:16 AM

CROSS REFERENCES

AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475,AL834187, AY358085; Mouse: AK089756, AY158090, AY506558: NP 112571.1;WO2003/024392 (claim 2, FIG. 97); Nakayama et al (2000) Biochem.Biophys. Res. Commun. 277(1):124-127; WO2003/077836; WO2001/38490 (claim3, FIG. 18B-1-18B-2).

(36) TENB2 (TMEFF2, Tomoregulin, TPEF, HPP1, TR, Putative Transmembrane35 Proteoglycan, Related to the EGF/Heregulin Family of Growth Factorsand Follistatin); 374 aa)

Nucleotide

Genbank accession no AF179274

Genbank version no. AF179274.2 GI:12280939

Genbank record update date: Mar. 11, 2010 01:05 AM

Polypeptide

Genbank accession no. AAD55776

Genbank version no. AAD55776.2 GI:12280940

Genbank record update date: Mar. 11, 2010 01:05 AM

CROSS REFERENCES

NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276;NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; AY358907, CAF85723,CQ782436; WO2004/074320; JP2004113151; WO2003/042661; WO2003/009814;EP1295944 (pages 69-70); WO2002/30268 (page 329); WO2001/90304;US2004/249130; US2004/022727; WO2004/063355; US2004/197325;US2003/232350; US2004/005563; US2003/124579; Horie et al (2000) Genomics67:146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun.266:593-602; Liang et al (2000) Cancer Res. 60:4907-12; Glynne-Jones etal (2001) Int J Cancer. October 15; 94(2):178-84.

(37) PSMA—FOLH1 (Folate Hydrolase (Prostate-Specific Membrane Antigen)1)

Nucleotide

Genbank accession no M99487

Genbank version no. M99487.1 GI:190663

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA60209

Genbank version no. AAA60209.1 GI:190664

Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

Israeli R. S., et al Cancer Res. 53 (2), 227-230 (1993)

Other Information

Official Symbol: FOLH1

Other Aliases: GIG27, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase, PSM,PSMA,

mGCP

Other Designations: N-acetylated alpha-linked acidic dipeptidase 1;N-acetylated-alpha-linked acidic dipeptidase I; NAALADase I; cellgrowth-inhibiting gene 27 protein; folylpoly-gamma-glutamatecarboxypeptidase; glutamate carboxylase II; glutamate carboxypeptidase2; glutamate carboxypeptidase II; membrane glutamate carboxypeptidase;prostate specific membrane antigen variant F;pteroylpoly-gamma-glutamate carboxypeptidase

Antibodies

U.S. Pat. No. 7,666,425:

Antibodies produces by Hybridomas having the following ATCC references:ATCC accession No. HB-12101, ATCC accession No. HB-12109, ATCC accessionNo. HB-12127 and ATCC accession No. HB-12126.

Proscan: a monoclonal antibody selected from the group consisting of8H12, 3E11, 17G1, 29B4, 30C1 and 20F2 (U.S. Pat. No. 7,811,564; MoffettS., et al Hybridoma (Larchmt). 2007 December; 26(6):363-72).

Cytogen: monoclonal antibodies 7E11-C5 (ATCC accession No. HB 10494) and9H10-A4 (ATCC accession No. HB11430)—U.S. Pat. No. 5,763,202GlycoMimetics: NUH2-ATCC accession No. HB 9762 (U.S. Pat. No. 7,135,301)Human Genome Science: HPRAJ70—ATCC accession No. 97131 (U.S. Pat. No.6,824,993); Amino acid sequence encoded by the cDNA clone (HPRAJ70)deposited as American Type Culture Collection (“ATCC”) Deposit No. 97131

Medarex: Anti-PSMA antibodies that lack fucosyl residues—U.S. Pat. No.7,875,278

Mouse anti-PSMA antibodies include the 3F5.4G6, 3D7.1.1, 4E10-1.14,3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C89, 3G6, 4C89, andmonoclonal antibodies. Hybridomas secreting 3F5.4G6, 3D7.1.1, 4E10-1.14,3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6 or 4C8B9have been publicly deposited and are described in U.S. Pat. No.6,159,508. Relevant hybridomas have been publicly deposited and aredescribed in U.S. Pat. No. 6,107,090. Moreover, humanized anti-PSMAantibodies, including a humanized version of J591, are described infurther detail in PCT Publication WO 02/098897.

Other mouse anti-human PSMA antibodies have been described in the art,such as mAb 107-1A4 (Wang, S. et al. (2001) Int. J. Cancer 92:871-876)and mAb 2C9 (Kato, K. et al. (2003) Int. J. Urol. 10:439-444).

Examples of human anti-PSMA monoclonal antibodies include the 4A3, 7F12,8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 antibodies, isolated andstructurally characterized as originally described in PCT PublicationsWO 01/09192 and WO 03/064606 and in U.S. Provisional Application Ser.No. 60/654,125, entitled “Human Monoclonal Antibodies to ProstateSpecific Membrane Antigen (PSMA)”, filed on Feb. 18, 2005. The V.sub.Hamino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and1C3 are shown in SEQ ID NOs: 1-9, respectively. The V.sub.L amino acidsequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 areshown in SEQ ID NOs: 10-18, respectively.

Other human anti-PSMA antibodies include the antibodies disclosed in PCTPublication WO 03/034903 and US Application No. 2004/0033229.

NW Biotherapeutics: A hybridoma cell line selected from the groupconsisting of 3F5.4G6 having ATCC accession number HB12060, 3D7-1.I.having ATCC accession number HB12309, 4E10-1.14 having ATCC accessionnumber HB12310, 3E11 (ATCC HB12488), 4D8 (ATCC HB12487), 3E6 (ATCCHB12486), 3C9 (ATCC HB12484), 2C7 (ATCC HB12490), 1G3 (ATCC HB12489),3C4 (ATCC HB12494), 3C6 (ATCC HB12491), 4D4 (ATCC HB12493), 1G9 (ATCCHB12495), 5C8B9 (ATCC HB12492) and 3G6 (ATCC HB12485)—see U.S. Pat. No.6,150,508

PSMA Development Company/Progenics/Cytogen—Seattle Genetics: mAb 3.9,produced by the hybridoma deposited under ATCC Accession No. PTA-3258 ormAb 10.3, produced by the hybridoma deposited under ATCC Accession No.PTA-3347—U.S. Pat. No. 7,850,971

PSMA Development Company—Compositions of PSMA antibodies (US20080286284, Table 1)

-   -   This application is a divisional of U.S. patent application Ser.        No. 10/395,894, filed on Mar. 21, 2003 (U.S. Pat. No. 7,850,971)

University Hospital Freiburg, Germany—mAbs 3/A12, 3/E7, and 3/F11 (WolfP., et al Prostate. 2010 Apr. 1; 70(5):562-9).

(38) SST (Somatostatin Receptor; Note that there are 5 Subtypes)

(38.1) SSTR2 (Somatostatin Receptor 2)

Nucleotide

Genbank accession no NM_001050

Genbank version no. NM_001050.2 GI:44890054

Genbank record update date: Aug. 19, 2012 01:37 PM

Polypeptide

Genbank accession no. NP_001041

Genbank version no. NP_001041.1 GI:4557859

Genbank record update date: Aug. 19, 2012 01:37 PM

CROSS REFERENCES

Yamada Y., et al Proc. Natl. Acad. Sci. U.S.A. 89 (1), 251-255 (1992);Susini C., et al Ann Oncol. 2006 December; 17(12):1733-42

Other Information

Official Symbol: SSTR2

Other Designations: SRIF-1; SS2R; somatostatin receptor type 2

(38.2) SSTR5 (Somatostatin Receptor 5)

Nucleotide

Genbank accession no D16827

Genbank version no. D16827.1 GI:487683

Genbank record update date: Aug. 1, 2006 12:45 PM

Polypeptide

Genbank accession no. BAA04107

Genbank version no. BAA04107.1 GI:487684

Genbank record update date: Aug. 1, 2006 12:45 PM

CROSS REFERENCES

Yamada, Y., et al Biochem. Biophys. Res. Commun. 195 (2), 844-852 (1993)

Other Information

Official Symbol: SSTR5

Other Aliases: SS-5-R

Other Designations: Somatostatin receptor subtype 5; somatostatinreceptor type 5

(38.3) SSTR1

(38.4) SSTR3

(38.5) SSTR4

AvB6—Both Subunits (39+40)

(39) ITGAV (Integrin, alpha V;

Nucleotide

Genbank accession no M14648 J02826 M18365

Genbank version no. M14648.1 GI:340306

Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA36808

Genbank version no. AAA36808.1 GI:340307

Genbank record update date: Jun. 23, 2010 08:56 AM

CROSS REFERENCES

Suzuki S., et al Proc. Natl. Acad. Sci. U.S.A. 83 (22), 8614-8618 (1986)

Other Information

Official Symbol: ITGAV

Other Aliases: CD51, MSK8, VNRA, VTNR

Other Designations: antigen identified by monoclonal antibody L230;integrin alpha-V; integrin alphaVbeta3; integrin, alpha V (vitronectinreceptor, alpha polypeptide, antigen CD51); vitronectin receptor subunitalpha (40) ITGB6 (Integrin, beta 6)

Nucleotide

Genbank accession no NM_000888

Genbank version no. NM_000888.3 GI:9966771

Genbank record update date: Jun. 27, 2012 12:46 AM

Polypeptide

Genbank accession no. NP_000879

Genbank version no. NP_000879.2 GI:9625002

Genbank record update date: Jun. 27, 2012 12:46 AM

CROSS REFERENCES

Sheppard D. J., et al Biol. Chem. 265 (20), 11502-11507 (1990)

Other Information

Official Symbol: ITGB6

Other Designations: integrin beta-6

Antibodies

Biogen: U.S. Pat. No. 7,943,742—Hybridoma clones 6.3G9 and 6.8G6 weredeposited with the ATCC, accession numbers ATCC PTA-3649 and -3645,respectively.

Biogen: U.S. Pat. No. 7,465,449—In some embodiments, the antibodycomprises the same heavy and light chain polypeptide sequences as anantibody produced by hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10,6.2A1, 6.2E5, 7.1G10, 7.7G5, or 7.1C5.

Centocor (J&J): U.S. Pat. Nos. 7,550,142; 7,163,681

-   -   For example in U.S. Pat. No. 7,550,142—an antibody having human        heavy chain and human light chain variable regions comprising        the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Seattle Genetics: 15H3 (Ryan MC., et al Cancer Res Apr. 15, 2012; 72(8Supplement): 4630)

(41) CEACAM5 (Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5)

Nucleotide

Genbank accession no M17303

Genbank version no. M17303.1 GI:178676

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAB59513

Genbank version no. AAB59513.1 GI:178677

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS REFERENCES

Beauchemin N., et al Mol. Cell. Biol. 7 (9), 3221-3230 (1987)

Other Information

Official Symbol: CEACAM5

Other Aliases: CD66e, CEA

Other Designations: meconium antigen 100

Antibodies

AstraZeneca-Medlmmune:US 20100330103; US20080057063;

-   -   US20020142359        -   for example an antibody having complementarity determining            regions (CDRs) with the following sequences: heavy chain;            CDR1—DNYMH, CDR2—WIDPENGDTE YAPKFRG, CDR3—LIYAGYLAMD Y; and            light chain CDR1—SASSSVTYMH, CDR2—STSNLAS, CDR3-QQRSTYPLT.        -   Hybridoma 806.077 deposited as European Collection of Cell            Cultures (ECACC) deposit no. 96022936.

Research Corporation Technologies, Inc.: U.S. Pat. No. 5,047,507

Bayer Corporation: U.S. Pat. No. 6,013,772

BioAlliance: U.S. Pat. Nos. 7,982,017; 7,674,605

-   -   U.S. Pat. No. 7,674,605        -   an antibody comprising the heavy chain variable region            sequence from the amino acid sequence of SEQ ID NO: 1, and            the light chain variable region sequence from the amino acid            sequence of SEQ ID NO:2.        -   an antibody comprising the heavy chain variable region            sequence from the amino acid sequence of SEQ ID NO:5, and            the light chain variable region sequence from the amino acid            sequence of SEQ ID NO:6.

Celltech Therapeutics Limited: U.S. Pat. No. 5,877,293

The Dow Chemical Company: U.S. Pat. Nos. 5,472,693; 6,417,337; 6,333,405

-   -   U.S. Pat. No. 5,472,693—for example, ATCC No. CRL-11215    -   U.S. Pat. No. 6,417,337—for example, ATCC CRL-12208    -   U.S. Pat. No. 6,333,405—for example, ATCC CRL-12208

Immunomedics, Inc: U.S. Pat. Nos. 7,534,431; 7,230,084; 7,300,644;6,730,300;

-   -   US20110189085        -   an antibody having CDRs of the light chain variable region            comprise: CDR1 comprises KASQDVGTSVA (SEQ ID NO: 20); CDR2            comprises WTSTRHT (SEQ ID NO: 21); and CDR3 comprises            QQYSLYRS (SEQ ID NO: 22);        -   and the CDRs of the heavy chain variable region of said            anti-CEA antibody comprise: CDR1 comprises TYWMS (SEQ ID NO:            23); CDR2 comprises EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and            CDR3 comprises LYFGFPWFAY (SEQ ID NO: 25).    -   US20100221175; US20090092598; US20070202044; US20110064653;        US20090185974; US20080069775.

(42) MET (Met Proto-Oncogene; Hepatocyte Growth Factor Receptor)

Nucleotide

Genbank accession no M35073

Genbank version no. M35073.1 GI:187553

Genbank record update date: Mar. 6, 2012 11:12 AM

Polypeptide

Genbank accession no. AAA59589

Genbank version no. AAA59589.1 GI:553531

Genbank record update date: Mar. 6, 2012 11:12 AM

CROSS REFERENCES

Dean M., et al Nature 318 (6044), 385-388 (1985)

Other Information

Official Symbol: MET

Other Aliases: AUTS9, HGFR, RCCP2, c-Met

Other Designations: HGF receptor; HGF/SF receptor; SF receptor;hepatocyte growth factor receptor; met proto-oncogene tyrosine kinase;proto-oncogene c-Met; scatter factor receptor; tyrosine-protein kinaseMet

Antibodies

Abgenix/Pfizer:US20100040629

-   -   for example, the antibody produced by hybridoma 13.3.2 having        American Type Culture Collection (ATCC) accession number        PTA-5026; the antibody produced by hybridoma 9.1.2 having ATCC        accession number PTA-5027; the antibody produced by hybridoma        8.70.2 having ATCC accession number PTA-5028; or the antibody        produced by hybridoma 6.90.3 having ATCC accession number        PTA-5029.

Amgen/Pfizer: US20050054019

-   -   for example, an antibody comprising a heavy chain having the        amino acid sequences set forth in SEQ ID NO: 2 where X2 is        glutamate and X4 is serine and a light chain having the amino        acid sequence set forth in SEQ ID NO: 4 where X8 is alanine,        without the signal sequences; an antibody comprising a heavy        chain having the amino acid sequences set forth in SEQ ID NO: 6        and a light chain having the amino acid sequence set forth in        SEQ ID NO: 8, without the signal sequences; an antibody        comprising a heavy chain having the amino acid sequences set        forth in SEQ ID NO: 10 and a light chain having the amino acid        sequence set forth in SEQ ID NO: 12, without the signal        sequences; or an antibody comprising a heavy chain having the        amino acid sequences set forth in SEQ ID NO: 14 and a light        chain having the amino acid sequence set forth in SEQ ID NO: 16,        without the signal sequences.

Agouron Pharmaceuticals (Now Pfizer): US20060035907

Eli Lilly: US20100129369

Genentech: U.S. Pat. No. 5,686,292; US20100028337; US20100016241;US20070129301; US20070098707; US20070092520, US20060270594;US20060134104; US20060035278; US20050233960; US20050037431

-   -   U.S. Pat. No. 5,686,292—for example, ATCC HB-11894 and ATCC        HB-11895    -   US 20100016241—for example, ATCC HB-11894 (hybridoma 1A3.3.13)        or HB-11895 (hybridoma 5D5.11.6)

National Defense Medical Center, Taiwan: Lu R M., et al Biomaterials.2011 April; 32(12):3265-74.

Novartis: US20090175860

-   -   for example, an antibody comprising the sequences of CDR1, CDR2        and CDR3 of heavy chain 4687, wherein the sequences of CDR1,        CDR2, and CDR3 of heavy chain 4687 are residues 26-35, 50-65,        and 98-102, respectively, of SEQ ID NO: 58; and the sequences of        CDR1, CDR2, and CDR3 of light chain 5097, wherein the sequences        of CDR1, CDR2, and CDR3 of light chain 5097 are residues 24-39,        55-61, and 94-100 of SEQ ID NO: 37.

Pharmacia Corporation: US20040166544

Pierre Fabre: US20110239316, US20110097262, US20100115639

Sumsung: US 20110129481—for example a monoclonal antibody produced froma hybridoma cell having accession number KCLRF-BP-00219 or accessionnumber of KCLRF-BP-00223.

Samsung: US 20110104176—for example an antibody produced by a hybridomacell having Accession Number: KCLRF-BP-00220.

University of Turin Medical School: DN-30 Pacchiana G., et al J BiolChem. 2010 Nov. 12; 285(46):36149-57

Van Andel Research Institute: Jiao Y., et al Mol Biotechnol. 2005September; 31(1):41-54.

(43) MUC1 (Mucin 1, Cell Surface Associated)

Nucleotide

Genbank accession no J05581

Genbank version no. J05581.1 GI:188869

Genbank record update date: Jun. 23, 2010 08:48 AM

Polypeptide

Genbank accession no. AAA59876

Genbank version no. AAA59876.1 GI:188870

Genbank record update date: Jun. 23, 2010 08:48 AM

CROSS REFERENCES

Gendler S. J., et al J. Biol. Chem. 265 (25), 15286-15293 (1990)

Other Information

Official Symbol: MUC1

Other Aliases: RP11-263K19.2, CD227, EMA, H23AG, KL-6, MAM6, MUC-1,MUC-1/SEC, MUC-1/X, MUC1/ZD, PEM, PEMT, PUM

Other Designations: DF3 antigen; H23 antigen; breastcarcinoma-associated antigen DF3; carcinoma-associated mucin; episialin;krebs von den Lungen-6; mucin 1, transmembrane; mucin-1; peanut-reactiveurinary mucin; polymorphic epithelial mucin; tumor associated epithelialmucin; tumor-associated epithelial membrane antigen; tumor-associatedmucin

Antibodies

AltaRex—Quest Pharma Tech: U.S. Pat. No. 6,716,966—for example an Alt-1antibody produced by the hybridoma ATCC No PTA-975.

AltaRex—Quest Pharma Tech: U.S. Pat. No. 7,147,850

CRT: 5E5—Sorensen A L., et al Glycobiology vol. 16 no. 2 pp. 96-107,2006; HMFG2-Burchell J., et al Cancer Res., 47, 5476-5482 (1987); seeWO2015/159076

Glycotope GT-MAB: GT-MAB 2.5-GEX (Website:

http://www.glycotope.com/pipeline/pankomab-gex)

Immunogen: U.S. Pat. No. 7,202,346

-   -   for example, antibody MJ-170: hybridoma cell line MJ-170 ATCC        accession no. PTA-5286Monoclonal antibody MJ-171: hybridoma cell        line MJ-171 ATCC accession no. PTA-5287; monoclonal antibody        MJ-172: hybridoma cell line MJ-172 ATCC accession no. PTA-5288;        or monoclonal antibody MJ-173: hybridoma cell line MJ-173 ATCC        accession no. PTA-5302

Immunomedics: U.S. Pat. No. 6,653,104

Ramot Tel Aviv Uni: U.S. Pat. No. 7,897,351

Regents Uni. CA: U.S. Pat. No. 7,183,388; US20040005647; US20030077676.

Roche GlycArt: U.S. Pat. No. 8,021,856

Russian National Cancer Research Center: Imuteran—Ivanov PK., et alBiotechnol J. 2007 July; 2(7):863-70

Technische Univ Braunschweig: (IIB6, HT186-B7, HT186-D11, HT186-G2,HT200-3A-C1, HT220-M-D1, HT220-M-G8)—Thie H., et al PLoS One. 2011 Jan.14; 6(1):e15921

(44) CA9 (Carbonic anhydrase IX)

Nucleotide

Genbank accession no. X66839

Genbank version no. X66839.1 GI:1000701

Genbank record update date: Feb. 2, 2011 10:15 AM

Polypeptide

Genbank accession no. CAA47315

Genbank version no. CAA47315.1 GI:1000702

Genbank record update date: Feb. 2, 2011 10:15 AM

CROSS REFERENCES

Pastorek J., et al Oncogene 9 (10), 2877-2888 (1994)

Other Information

Official Symbol: CA9

Other Aliases: CAIX, MN

Other Designations: CA-IX; P54/58N; RCC-associated antigen G250;RCC-associated protein G250; carbonate dehydratase IX; carbonicanhydrase 9; carbonic dehydratase; membrane antigen MN; pMW1; renal cellcarcinoma-associated antigen G250

Antibodies

Abgenix/Amgen:US20040018198

Affibody: Anti-CAIX Affibody molecules

-   -   (http://www.affibody.com/en/Product-Portfolio/Pipeline/)

Bayer: U.S. Pat. No. 7,462,696

Bayer/Morphosys: 3ee9 mAb—Petrul H M., et al Mol Cancer Ther. 2012February; 11(2):340-9

Harvard Medical School: Antibodies G10, G36, G37, G39, G45, G57, G106,G119, G6, G27, G40 and G125. Xu C., et al PLoS One. 2010 Mar. 10;5(3):e9625

Institute of Virology, Slovak Academy of Sciences (Bayer)—U.S. Pat. No.5,955,075

-   -   for example, M75—ATCC Accession No. HB 11128 or MN12—ATCC        Accession No. HB 11647 Institute of Virology, Slovak Academy of        Sciences: U.S. Pat. No. 7,816,493    -   for example the M75 monoclonal antibody that is secreted from        the hybridoma VU-M75, which was deposited at the American Type        Culture Collection under ATCC No. HB 11128; or the V/10        monoclonal antibody secreted from the hybridoma V/10-VU, which        was deposited at the International Depository Authority of the        Belgian Coordinated Collection of Microorganisms (BCCM) at the        Laboratorium voor Moleculaire Bioloqie-Plasmidencollectie (LMBP)        at the Universeit Gent in Gent, Belgium, under Accession No.        LMBP 6009CB.

Institute of Virology, Slovak Academy of Sciences US20080177046;US20080176310; US20080176258; US20050031623

Novartis: US20090252738

Wilex: U.S. Pat. No. 7,691,375—for example the antibody produced by thehybridoma cell line DSM ASC 2526.

Wilex: US20110123537; Rencarex: Kennett RH., et al Curr Opin Mol Ther.2003 February; 5(1):70-5

Xencor: US20090162382

(45) EGFRvIII (Epidermal Growth Factor Receptor (EGFR), TranscriptVariant 3,

Nucleotide

Genbank accession no. NM_201283

Genbank version no. NM_201283.1 GI:41327733

Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_958440

Genbank version no. NP_958440.1 GI:41327734

Genbank record update date: Sep. 30, 2012 01:47 PM

CROSS-REFERENCES

Batra S K., et al Cell Growth Differ 1995; 6:1251-1259.

Antibodies:

U.S. Pat. Nos. 7,628,986 and 7,736,644 (Amgen)

-   -   For example, a heavy chain variable region amino acid sequence        selected from the group consisting of SEQ ID NO: 142 and        variants & a light chain variable region amino acid sequence        selected from the group consisting of: SEQ ID NO: 144 and        variants.

US20100111979 (Amgen)

-   -   For example, an antibody comprising a heavy chain amino acid        sequence comprising:    -   CDR1 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR1 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2),170 (SEQ ID NO:        4),150 (SEQ ID NO: 5),095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9),139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12),124 (SEQ ID NO: 13),318        (SEQ ID NO: 15),342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);        CDR2 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR2 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2),170 (SEQ ID NO:        4),150 (SEQ ID NO: 5),095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9),139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12),124 (SEQ ID NO: 13),318        (SEQ ID NO: 15),342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);        and    -   CDR3 consisting of a sequence selected from the group consisting        of the amino acid sequences for the CDR3 region of antibodies        13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2),170 (SEQ ID NO:        4),150 (SEQ ID NO: 5),095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9),139        (SEQ ID NO: 10), 211 (SEQ ID NO: 12),124 (SEQ ID NO: 13),318        (SEQ ID NO: 15),342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17).

US20090240038 (Amgen)

-   -   For example, an antibody having at least one of the heavy or        light chain polypeptides comprises an amino acid sequence that        is at least 90% identical to the amino acid sequence selected        from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ        ID NO: 142, SEQ ID NO: 144, and any combination thereof.

US20090175887 (Amgen)

-   -   For example, an antibody having a heavy chain amino acid        sequence selected from the group consisting of the heavy chain        amino acid sequence of antibody 13.1.2 (SEQ ID NO: 138), 131        (SEQ ID NO: 2),170 (SEQ ID NO: 4),150 (SEQ ID NO: 5), 095 (SEQ        ID NO: 7),250 (SEQ ID NO: 9),139 (SEQ ID NO: 10), 211 (SEQ ID        NO: 12),124 (SEQ ID NO: 13),318 (SEQ ID NO: 15),342 (SEQ ID NO:        16), and 333 (SEQ ID NO: 17).

US20090156790 (Amgen)

-   -   For example, antibody having heavy chain polypeptide and a light        chain polypeptide, wherein at least one of the heavy or light        chain polypeptides comprises an amino acid sequence that is at        least 90% identical to the amino acid sequence selected from the        group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO:        142, SEQ ID NO: 144, and any combination thereof.

US20090155282, US20050059087 and US20050053608 (Amgen)

-   -   For example, an antibody heavy chain amino acid sequence        selected from the group consisting of the heavy chain amino acid        sequence of antibody 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO:        2),170 (SEQ ID NO: 4),150 (SEQ ID NO: 5),095 (SEQ ID NO: 7),250        (SEQ ID NO: 9),139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124        (SEQ ID NO: 13),318 (SEQ ID NO: 15),342 (SEQ ID NO: 16), and 333        (SEQ ID NO: 17).

MR1-1 (U.S. Pat. No. 7,129,332; Duke)

-   -   For example, a variant antibody having the sequence of SEQ ID        NO.18 with the substitutions S98P-T99Y in the CDR3 VH, and F92W        in CDR3 VL.

L8A4, H10, Y10 (Wikstrand CJ., et al Cancer Res. 1995 Jul. 15;55(14):3140-8; Duke)

US20090311803 (Harvard University)

-   -   For example, SEQ ID NO:9 for antibody heavy chain variable        region, and SEQ ID NO: 3 for light chain variable region amino        acid sequences

US20070274991 (EMD72000, also known as matuzumab; Harvard University)

-   -   For example, SEQ ID NOs: 3 & 9 for light chain and heavy chain        respectively

U.S. Pat. No. 6,129,915 (Schering)

-   -   For example, SEQ. ID NOs: 1, 2, 3, 4, 5 and 6.

mAb CH12—Wang H., et al FASEB J. 2012 January; 26(1):73-80 (ShanghaiCancer Institute).

RAbDMvIII—Gupta P., et al BMC Biotechnol. 2010 Oct. 7; 10:72 (StanfordUniversity Medical Center).

mAb Ua30—Ohman L., et al Tumour Biol. 2002 March-April; 23(2):61-9(Uppsala University).

Han D G., et al Nan Fang Yi Ke Da Xue Xue Bao. 2010 January; 30(1):25-9(Xi'an Jiaotong University).

(46) CD33 (CD33 Molecule)

Nucleotide

Genbank accession no. M_23197

Genbank version no. NM_23197.1 GI:180097

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA51948

Genbank version no. AAA51948.1 GI:188098

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS-REFERENCES

Simmons D., et al J. Immunol. 141 (8), 2797-2800 (1988)

Other Information

Official Symbol: CD33

Other Aliases: SIGLEC-3, SIGLEC3, p67

Other Designations: CD33 antigen (gp67); gp67; myeloid cell surfaceantigen CD33; sialic acid binding Ig-like lectin 3; sialic acid-bindingIg-like lectin

Antibodies

H195 (Lintuzumab)—Raza A., et al Leuk Lymphoma. 2009 August;50(8):1336-44; U.S. Pat. No. 6,759,045 (Seattle Genetics/Immunomedics)

mAb OKT9: Sutherland, D. R. et al. Proc Natl Acad Sci USA 78(7):4515-4519 1981, Schneider, C., et al J Biol Chem 257, 8516-8522 (1982)

mAb E6: Hoogenboom, H. R., et al J Immunol 144, 3211-3217 (1990)

U.S. Pat. No. 6,590,088 (Human Genome Sciences)

-   -   For example, SEQ ID NOs: 1 and 2 and ATCC accession no. 97521

U.S. Pat. No. 7,557,189 (Immunogen)

-   -   For example, an antibody or fragment thereof comprising a heavy        chain variable region which comprises three CDRs having the        amino acid sequences of SEQ ID NOs:1-3 and a light chain        variable region comprising three CDRs having the amino acid        sequences of SEQ ID NOs:4-6.

(47) CD19 (CD19 Molecule)

Nucleotide

Genbank accession no. NM_001178098

Genbank version no. NM_001178098.1 GI:296010920

Genbank record update date: Sep. 10, 2012 12:43 AM

Polypeptide

Genbank accession no. NP_001171569

Genbank version no. NP_001171569.1 GI:296010921

Genbank record update date: Sep. 10, 2012 12:43 AM

CROSS-REFERENCES

Tedder T F., et al J. Immunol. 143 (2): 712-7 (1989)

Other Information

Official Symbol: CD19

Other Aliases: B4, CVID3

Other Designations: B-lymphocyte antigen CD19; B-lymphocyte surfaceantigen B4; T-cell surface antigen Leu-12; differentiation antigen CD19

Antibodies

Immunogen: HuB4—A-Katib AM., et al Clin Cancer Res. 2009 Jun. 15;15(12):4038-45.

4G7: Kügler M., et al Protein Eng Des Sel. 2009 March; 22(3):135-47

-   -   For example, sequences in FIG. 3 of of Knappik, A. et al. J Mol        Biol 2000 February; 296(1):57-86

AstraZeneca/MedImmune: MEDI-551—Herbst R., et al J Pharmacol Exp Ther.2010 October; 335(1):213-22

Glenmark Pharmaceuticals: GBR-401—Hou S., et al Mol Cancer Ther November2011 (Meeting Abstract Supplement) C164

U.S. Pat. No. 7,109,304 (Immunomedics)

-   -   For example, an antibody comprising the sequence of hA19Vk (SEQ        ID NO:7) and the sequence of hA19VH (SEQ ID NO:10)

U.S. Pat. No. 7,902,338 (Immunomedics)

-   -   For example, an antibody or antigen-binding fragment thereof        that comprises the light chain complementarity determining        region CDR sequences CDR1 of SEQ ID NO: 16 (KASQSVDYDGDSYLN);        CDR2 of SEQ ID NO: 17 (DASNLVS); and CDR3 of SEQ ID NO: 18        (QQSTEDPWT) and the heavy chain CDR sequences CDR1 of SEQ ID NO:        19 (SYWMN); CDR2 of SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3        of SEQ ID NO: 21 (RETTTVGRYYYAMDY) and also comprises human        antibody framework (FR) and constant region sequences with one        or more framework region amino acid residues substituted from        the corresponding framework region sequences of the parent        murine antibody, and wherein said substituted FR residues        comprise the substitution of serine for phenylalanine at Kabat        residue 91 of the heavy chain variable region.

Medarex: MDX-1342—Cardarelli P M., et al Cancer Immunol Immunother. 2010February; 59(2):257-65.

MorphoSys/Xencor: MOR-208/XmAb-5574—Zalevsky J., et al Blood. 2009 Apr.16; 113(16):3735-43

U.S. Pat. No. 7,968,687 (Seattle Genetics)

-   -   An antibody or antigen-binding fragment comprising a heavy chain        variable domain comprising the amino acid sequence of SEQ ID        NO:9 and a light chain variable domain comprising the amino acid        sequence of SEQ ID NO: 24.

4G7 chim—Lang P., et al Blood. 2004 May 15; 103(10):3982-5 (Universityof Tubingen)

-   -   For example, FIG. 6 and SEQ ID No: 80 of US20120082664

Zhejiang University School of Medicine: 2E8—Zhang J., et al J DrugTarget. 2010 November; 18(9):675-8

(48) IL2RA (Interleukin 2 Receptor, Alpha); NCBI Reference Sequence:NM_000417.2);

Nucleotide

Genbank accession no. NM_000417

Genbank version no. NM_000417.2 GI:269973860

Genbank record update date: Sep. 9, 2012 04:59 PM

Polypeptide

Genbank accession no. NP_000408

Genbank version no. NP_000408.1 GI:4557667

Genbank record update date: Sep. 9, 2012 04:59 PM

CROSS-REFERENCES

Kuziel W. A., et al J. Invest. Dermatol. 94 (6 SUPPL), 27S-32S (1990)

Other Information

Official Symbol: IL2RA

Other Aliases: RP11-536K7.1, CD25, IDDM10, IL2R, TCGFR

Other Designations: FIL-2 receptor subunit alpha; IL-2-RA; IL-2R subunitalpha; 1L2-RA; TAC antigen; interleukin-2 receptor subunit alpha; p55

Antibodies

U.S. Pat. No. 6,383,487 (Novartis/UCL: Baxilisimab [Simulect])

U.S. Pat. No. 6,521,230 (Novartis/UCL: Baxilisimab [Simulect])

-   -   For example, an antibody having an antigen binding site        comprises at least one domain which comprises CDR1 having the        amino acid sequence in SEQ. ID. NO: 7, CDR2 having the amino        acid sequence in SEQ. ID. NO: 8, and CDR3 having the amino acid        sequence in SEQ. ID. NO: 9; or said CDR1, CDR2 and CDR3 taken in        sequence as a whole comprise an amino acid sequence which is at        least 90% identical to SEQ. ID. NOs: 7, 8 and 9 taken in        sequence as a whole.

Daclizumab—Rech A J., et al Ann NY Acad Sci. 2009 September; 1174:99-106(Roche)

(49) AXL (AXl Receptor Tyrosine Kinase)

Nucleotide

Genbank accession no. M76125

Genbank version no. M76125.1 GI:292869

Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA61243

Genbank version no. AAA61243.1 GI:29870

Genbank record update date: Jun. 23, 2010 08:53 AM

CROSS-REFERENCES

O'Bryan J. P., et al Mol. Cell. Biol. 11 (10), 5016-5031 (1991);Bergsagel P. L., et al J. Immunol. 148 (2), 590-596 (1992)

Other Information

Official Symbol: AXL

Other Aliases: JTK11, UFO

Other Designations: AXL oncogene; AXL transforming sequence/gene;oncogene AXL; tyrosine-protein kinase receptor UFO

Antibodies

YW327.6S2—Ye X., et al Oncogene. 2010 Sep. 23; 29(38):5254-64.(Genentech)

BergenBio: BGB324 (http://www.bergenbio.com/BGB324)

(50) CD30—TNFRSF8 (Tumor Necrosis Factor Receptor Superfamily, Member 8)

Nucleotide

Genbank accession no. M83554

Genbank version no. M83554.1 GI:180095

Genbank record update date: Jun. 23, 2010 08:53 AM

Polypeptide

Genbank accession no. AAA51947

Genbank version no. AAA51947.1 GI:180096

Genbank record update date: Jun. 23, 2010 08:53 AM

CROSS-REFERENCES

Durkop H., et al Cell 68 (3), 421-427 (1992)

Other Information

Official Symbol: TNFRSF8

Other Aliases: CD30, D1S166E, Ki-1

Other Designations: CD30L receptor; Ki-1 antigen; cytokine receptorCD30; lymphocyte activation antigen CD30; tumor necrosis factor receptorsuperfamily member 8

(51) BCMA (B-Cell Maturation Antigen)—TNFRSF17 (Tumor Necrosis FactorReceptor Superfamily, Member 17)

Nucleotide

Genbank accession no. Z29574

Genbank version no. Z29574.1 GI:471244

Genbank record update date: Feb. 2, 2011 10:40 AM

Polypeptide

Genbank accession no. CAA82690

Genbank version no. CAA82690.1 GI:471245

Genbank record update date: Feb. 2, 2011 10:40 AM

CROSS-REFERENCES

Laabi Y., et al Nucleic Acids Res. 22 (7), 1147-1154 (1994)

Other Information

Official Symbol: TNFRSF17

Other Aliases: BCM, BCMA, CD269

Other Designations: B cell maturation antigen; B-cell maturation factor;B-cell maturation protein; tumor necrosis factor receptor superfamilymember 17

(52) CT Ags—CTA (Cancer Testis Antigens)

CROSS-REFERENCES

Fratta E., et al. Mol Oncol. 2011 April; 5(2):164-82; Lim S H., at al AmJ Blood Res. 2012; 2(1):29-35.

(53) CD174 (Lewis Y)—FUT3 (Fucosyltransferase 3 (Galactoside3(4)-L-Fucosyltransferase, Lewis blood group)

Nucleotide

Genbank accession no. NM000149

Genbank version no. NM000149.3 GI:148277008

Genbank record update date: Jun. 26, 2012 04:49 PM

Polypeptide

Genbank accession no. NP_000140

Genbank version no. NP_000140.1 GI:4503809

Genbank record update date: Jun. 26, 2012 04:49 PM

CROSS-REFERENCES

Kukowska-Latallo, J. F., et al Genes Dev. 4 (8), 1288-1303 (1990)

Other Information

Official Symbol: FUT3

Other Aliases: CD174, FT3B, FucT-Ill, LE, Les

Other Designations: Lewis FT; alpha-(1,3/1,4)-fucosyltransferase; bloodgroup Lewis alpha-4-fucosyltransferase; fucosyltransferase Ill;galactoside 3(4)-L-fucosyltransferase

(54) CLEC14A (C-Type Lectin Domain Family 14, Member a; GenbankAccession No. NM175060)

Nucleotide

Genbank accession no. NM175060

Genbank version no. NM175060.2 GI:371123930

Genbank record update date: Apr. 1, 2012 03:34 PM

Polypeptide

Genbank accession no. NP_778230

Genbank version no. NP_778230.1 GI:28269707

Genbank record update date: Apr. 1, 2012 03:34 PM

Other Information

Official Symbol: CLEC14A

Other Aliases: UNQ236/PRO269, C14orf27, CEG1, EGFR-5

Other Designations: C-type lectin domain family 14 member A; CIECT andEGF-like domain containing protein; epidermal growth factor receptor 5

(55) GRP78—HSPA5 (Heat Shock 70 kDa Protein 5 (Glucose-RegulatedProtein, 78 kDa)

Nucleotide

Genbank accession no. NM005347

Genbank version no. NM005347.4 GI:305855105

Genbank record update date: Sep. 30, 2012 01:42 PM

Polypeptide

Genbank accession no. NP_005338

Genbank version no. NP_005338.1 GI:16507237

Genbank record update date: Sep. 30, 2012 01:42 PM

CROSS-REFERENCES

Ting J., et al DNA 7 (4), 275-286 (1988)

Other Information

Official Symbol: HSPA5

Other Aliases: BIP, GRP78, MIF2

Other Designations: 78 kDa glucose-regulated protein; endoplasmicreticulum lumenal Ca(2+)-binding protein grp78; immunoglobulin heavychain-binding protein

(56) CD70 (CD70 Molecule) L08096

Nucleotide

Genbank accession no. L08096

Genbank version no. L08096.1 GI:307127

Genbank record update date: Jun. 23, 2012 08:54 AM

Polypeptide

Genbank accession no. AAA36175

Genbank version no. AAA36175.1 GI:307128

Genbank record update date: Jun. 23, 2012 08:54 AM

CROSS-REFERENCES

Goodwin R. G., et al Cell 73 (3), 447-456 (1993)

Other Information

Official Symbol: CD70

Other Aliases: CD27L, CD27LG, TNFSF7

Other Designations: CD27 ligand; CD27-L; CD70 antigen; Ki-24 antigen;surface antigen CD70; tumor necrosis factor (ligand) superfamily, member7; tumor necrosis factor ligand superfamily member 7

Antibodies

MDX-1411 against CD70 (Medarex)

-   h1F6 (Oflazoglu, E., et al, Clin Cancer Res. 2008 Oct. 1;    14(19):6171-80; Seattle Genetics)    -   For example, see US20060083736 SEQ ID NOs: 1, 2, 11 and 12 and        FIG. 1.

(57) Stem Cell Specific Antigens. For Example:

-   -   5T4 (see entry (63) below)    -   CD25 (see entry (48) above)    -   CD32        -   Polypeptide            -   Genbank accession no. ABK42161            -   Genbank version no. ABK42161.1 GI:117616286            -   Genbank record update date: Jul. 25, 2007 03:00 PM    -   LGR5/GPR49        -   Nucleotide            -   Genbank accession no. NM_003667            -   Genbank version no. NM_003667.2 GI:24475886            -   Genbank record update date: Jul. 22, 2012 03:38 PM        -   Polypeptide            -   Genbank accession no. NP_003658            -   Genbank version no. NP_003658.1 GI:4504379            -   Genbank record update date: Jul. 22, 2012 03:38 PM    -   Prominin/CD133        -   Nucleotide            -   Genbank accession no. NM_006017            -   Genbank version no. NM_006017.2 GI:224994187            -   Genbank record update date: Sep. 30, 2012 01:47 PM        -   Polypeptide            -   Genbank accession no. NP_006008            -   Genbank version no. NP_006008.1 GI:5174387            -   Genbank record update date: Sep. 30, 2012 01:47 PM

(58) ASG-5

CROSS-REFERENCES

(Smith L. M., et. al AACR 2010 Annual Meeting (abstract #2590); Gudas J.M., et. al. AACR 2010 Annual Meeting (abstract #4393)

Antibodies

Anti—AGS-5 Antibody: M6.131 (Smith, L. M., et. al AACR 2010 AnnualMeeting (abstract #2590)

(59) ENPP3 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 3)

Nucleotide

Genbank accession no. AF005632

Genbank version no. AF005632.2 GI:4432589

Genbank record update date: Mar. 10, 2010 09:41 PM

Polypeptide

Genbank accession no. AAC51813

Genbank version no. AAC51813.1 GI:2465540

Genbank record update date: Mar. 10, 2010 09:41 PM

CROSS-REFERENCES

Jin-Hua P., et al Genomics 45 (2), 412-415 (1997)

Other Information

Official Symbol: ENPP3

Other Aliases: RP5-988G15.3, B10, CD203c, NPP3, PD-IBETA, PDNP3

Other Designations: E-NPP 3; dJ1005H11.3 (phosphodiesterase 1/nucleotidepyrophosphatase 3); dJ914N13.3 (phosphodiesterase 1/nucleotidepyrophosphatase 3); ectonucleotide pyrophosphatase/phosphodiesterasefamily member 3; gpl30RB13-6; phosphodiesterase I beta;phosphodiesterase 1/nucleotide pyrophosphatase 3; phosphodiesterase-Ibeta

(60) PRR4 (Proline Rich 4 (Lacrimal))

Nucleotide

Genbank accession no. NM_007244

Genbank version no. NM_007244.2 GI:154448885

Genbank record update date: Jun. 28, 2012 12:39 PM

Polypeptide

Genbank accession no. NP_009175

Genbank version no. NP_009175.2 GI:154448886

Genbank record update date: Jun. 28, 2012 12:39 PM

CROSS-REFERENCES

Dickinson D. P., et al Invest. Ophthalmol. Vis. Sci. 36 (10), 2020-2031(1995)

Other Information

Official Symbol: PRR4

Other Aliases: LPRP, PROL4

Other Designations: lacrimal proline-rich protein; nasopharyngealcarcinoma-associated proline-rich protein 4; proline-rich polypeptide 4;proline-rich protein 4

(61) GCC—GUCY2C (Guanylate Cyclase 2C (Heat Stable Enterotoxin Receptor)

Nucleotide

Genbank accession no. NM_004963

Genbank version no. NM_004963.3 GI:222080082

Genbank record update date: Sep. 2, 2012 01:50 PM

Polypeptide

Genbank accession no. NP_004954

Genbank version no. NP_004954.2 GI:222080083

Genbank record update date: Sep. 2, 2012 01:50 PM

CROSS-REFERENCES

De Sauvage F. J., et al J. Biol. Chem. 266 (27), 17912-17918 (1991);Singh S., et al Biochem. Biophys. Res. Commun. 179 (3),1455-1463 (1991)

Other Information

Official Symbol: GUCY2C

Other Aliases: DIAR6, GUC2C, MUCIL, STAR

Other Designations: GC-C; STA receptor; guanylyl cyclase C; hSTAR;heat-stable enterotoxin receptor; intestinal guanylate cyclase

(62) Liv-1—SLC39A6 (Solute Carrier Family 39 (Zinc Transporter), Member6)

Nucleotide

Genbank accession no. U41060

Genbank version no. U41060.2 GI:12711792

Genbank record update date: Nov. 30, 2009 04:35 PM

Polypeptide

Genbank accession no. AAA96258

Genbank version no. AAA96258.2 GI:12711793

Genbank record update date: Nov. 30, 2009 04:35 PM

CROSS-REFERENCES

Taylor K M., et al Biochim Biophys Acta. 2003 Apr. 1; 1611(1-2):16-30

Other Information

Official Symbol: SLC39A6

Other Aliases: LIV-1

Other Designations: LIV-1 protein, estrogen regulated; ZIP-6;estrogen-regulated protein LIV-1; solute carrier family 39 (metal iontransporter), member 6; solute carrier family 39 member 6; zinctransporter ZIP6; zrt- and Irt-like protein 6

(63) 5T4, Trophoblast Glycoprotein, TPBG—TPBG (Trophoblast Glycoprotein)

Nucleotide

Genbank accession no. AJ012159

Genbank version no. AJ012159.1 GI:3805946

Genbank record update date: Feb. 1, 2011 10:27 AM

Polypeptide

Genbank accession no. CAA09930

Genbank version no. CAA09930.1 GI:3805947

Genbank record update date: Feb. 1, 2011 10:27 AM

CROSS-REFERENCES

King K. W., et al Biochim. Biophys. Acta 1445 (3), 257-270 (1999)

Other Information

Official Symbol: TPBG

“Other Aliases: 5T4, 5T4AG, M6P1

“Other Designations: 5T4 oncofetal antigen; 5T4 oncofetal trophoblastglycoprotein; 5T4 oncotrophoblast glycoprotein “See WO2015/155345

(64) CD56—NCMA1 (Neural cell adhesion molecule 1)

Nucleotide

Genbank accession no. NM_000615

Genbank version no. NM_000615.6 GI:336285433

Genbank record update date: Sep. 23, 2012 02:32 PM

Polypeptide

Genbank accession no. NP_000606

Genbank version no. NP_000606.3 GI:94420689

Genbank record update date: Sep. 23, 2012 02:32 PM

CROSS-REFERENCES

Dickson, G., et al, Cell 50 (7), 1119-1130 (1987)

Other Information

Official Symbol: NCAM1

Other Aliases: CD56, MSK39, NCAM

Other Designations: antigen recognized by monoclonal antibody 5.1H11;neural cell adhesion molecule, NCAM

Antibodies

Immunogen: HuN901 (Smith SV., et al Curr Opin Mol Ther. 2005 August;7(4):394-401)

-   -   For example, see humanized from murine N901 antibody. See FIGS.        1b and 1e of Roguska, M. A., et al. Proc Natl Acad Sci USA        February 1994; 91:969-973.

(65) CanAg (Tumor Associated Antigen CA242)

CROSS-REFERENCES

Haglund C., et al Br J Cancer 60:845-851, 1989; Baeckstrom D., et al JBiol Chem 266:21537-21547,1991

Antibodies

huC242 (Tolcher A W et al., J Clin Oncol. 2003 Jan. 15; 21(2):211-22;Immunogen)

-   -   For example, see US20080138898A1 SEQ ID NO: 1 and 2 (66) FOLR1        (Folate Receptor 1)

Nucleotide

Genbank accession no. J05013

Genbank version no. J05013.1 GI:182417

Genbank record update date: Jun. 23, 2010 08:47 AM

Polypeptide

Genbank accession no. AAA35823

Genbank version no. AAA35823.1 GI:182418

Genbank record update date: Jun. 23, 2010 08:47 AM

CROSS-REFERENCES

Elwood P. C., et al J. Biol. Chem. 264 (25), 14893-14901 (1989)

Other Information

Official Symbol: FOLR1

Other Aliases: FBP, FOLR

Other Designations: FR-alpha; KB cells FBP; adult folate-bindingprotein; folate binding protein; folate receptor alpha; folate receptor,adult; ovarian tumor-associated antigen MOv18

Antibodies

M9346A—Whiteman K R., et al Cancer Res Apr. 15, 2012; 72(8 Supplement):4628 (Immunogen)

(67) GPNMB (Glycoprotein (Transmembrane) Nmb)

Nucleotide

Genbank accession no. X76534

Genbank version no. X76534.1 GI:666042

Genbank record update date: Feb. 2, 2011 10:10 AM

Polypeptide

Genbank accession no. CAA54044

Genbank version no. CAA54044.1 GI:666043

Genbank record update date: Feb. 2, 2011 10:10 AM

CROSS-REFERENCES

Weterman M. A., et al Int. J. Cancer 60 (1), 73-81 (1995)

Other Information

Official Symbol: GPNMB

Other Aliases: UNQ1725/PRO9925, HGFIN, NMB

Other Designations: glycoprotein NMB; glycoprotein nmb-like protein;osteoactivin; transmembrane glycoprotein HGFIN; transmembraneglycoprotein NMB

Antibodies

Celldex Therapeutics: CR011 (Tse K F., et al Clin Cancer Res. 2006 Feb.15; 12(4):1373-82)

-   -   For example, see EP1827492B1 SEQ ID NO: 22, 24, 26, 31, 33 and        35

(68) TIM-1—HAVCR1 (Hepatitis a Virus Cellular Receptor 1)

Nucleotide

Genbank accession no. AF043724

Genbank version no. AF043724.1 GI:2827453

Genbank record update date: Mar. 10, 2010 06:24 PM

Polypeptide

Genbank accession no. AAC39862

Genbank version no. AAC39862.1 GI:2827454

Genbank record update date: Mar. 10, 2010 06:24 PM

CROSS-REFERENCES

Feigelstock D., et al J. Virol. 72 (8), 6621-6628 (1998)

Other Information

Official Symbol: HAVCR1

Other Aliases: HAVCR, HAVCR-1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1,TIMD1

Other Designations: T cell immunoglobin domain and mucin domain protein1; T-cell membrane protein 1; kidney injury molecule 1

(69) RG-1/Prostate Tumor Target Mindin—Mindin/RG-1

CROSS-REFERENCES

Parry R., et al Cancer Res. 2005 Sep. 15; 65(18):8397-405 (70)B7-H4—VTCN1 (V-set domain containing T cell activation inhibitor 1

Nucleotide

Genbank accession no. BX648021

Genbank version no. BX648021.1 GI:34367180

Genbank record update date: Feb. 2, 2011 08:40 AM

CROSS-REFERENCES

Sica G L., et al Immunity. 2003 June; 18(6):849-61

Other Information

Official Symbol: VTCN1

Other Aliases: RP11-229A19.4, B7-H4, B7H4, B7S1, B7X, B7h.5, PRO1291,VCTN1

Other Designations: B7 family member, H4; B7 superfamily member 1; Tcell costimulatory molecule B7x; T-cell costimulatory molecule B7x;V-set domain-containing T-cell activation inhibitor 1; immunecostimulatory protein B7-H4

(71) PTK7 (PTK7 Protein Tyrosine Kinase 7)

Nucleotide

Genbank accession no. AF447176

Genbank version no. AF447176.1 GI:17432420

Genbank record update date: Nov. 28, 2008 01:51 PM

Polypeptide

Genbank accession no. AAL39062

Genbank version no. AAL39062.1 GI:17432421

Genbank record update date: Nov. 28, 2008 01:51 PM

CROSS-REFERENCES

Park S. K., et al J. Biochem. 119 (2), 235-239 (1996)

Other Information

Official Symbol: PTK7

Other Aliases: CCK-4, CCK4

Other Designations: colon carcinoma kinase 4; inactive tyrosine-proteinkinase 7; pseudo tyrosine kinase receptor 7; tyrosine-proteinkinase-like 7

(72) CD37 (CD37 Molecule)

Nucleotide

Genbank accession no. NM_001040031

Genbank version no. NM_001040031.1 GI:91807109

Genbank record update date: Jul. 29, 2012 02:08 PM

Polypeptide

Genbank accession no. NP_001035120

Genbank version no. NP_001035120.1 GI:91807110

Genbank record update date: Jul. 29, 2012 02:08 PM

CROSS-REFERENCES

Schwartz-Albiez R., et al J. Immunol. 140 (3), 905-914 (1988)

Other Information

Official Symbol: CD37

Other Aliases: GP52-40, TSPAN26

Other Designations: CD37 antigen; cell differentiation antigen 37;leukocyte antigen CD37; leukocyte surface antigen CD37; tetraspanin-26;tspan-26

Antibodies

Boehringer Ingelheim: mAb 37.1 (Heider K H., et al Blood. 2011 Oct. 13;118(15):4159-68)

Trubion: CD37-SMIP (G28-1 scFv-Ig) ((Zhao X., et al Blood. 2007; 110:2569-2577)

-   -   For example, see US20110171208A1 SEQ ID NO: 253

Immunogen: K7153A (Deckert J., et al Cancer Res Apr. 15, 2012; 72(8Supplement): 4625)

(73) CD138—SDC1 (Syndecan 1)

Nucleotide

Genbank accession no. AJ551176

Genbank version no. AJ551176.1 GI:29243141

Genbank record update date: Feb. 1, 2011 12:09 PM

Polypeptide

Genbank accession no. CAD80245

Genbank version no. CAD80245.1 GI:29243142

Genbank record update date: Feb. 1, 2011 12:09 PM

CROSS-REFERENCES

O'Connell F P., et al Am J Clin Pathol. 2004 February; 121(2):254-63

Other Information

Official Symbol: SDC1

Other Aliases: CD138, SDC, SYND1, syndecan

Other Designations: CD138 antigen; heparan sulfate proteoglycanfibroblast growth factor receptor; syndecan proteoglycan 1; syndecan-1

Antibodies

Biotest: chimerized MAb (nBT062)—(Jagannath S., et al PosterASH #3060,2010; WIPO Patent Application WO/2010/128087)

-   -   For example, see US20090232810 SEQ ID NO: 1 and 2

Immunogen: B-B4 (Tassone P., et al Blood 104_3688-3696)

-   -   For example, see US20090175863A1 SEQ ID NO: 1 and 2

(74) CD74 (CD74 Molecule, Major Histocompatibility Complex, Class IIInvariant Chain)

Nucleotide

Genbank accession no. NM_004355

Genbank version no. NM_004355.1 GI:343403784

Genbank record update date: Sep. 23, 2012 02:30 PM

Polypeptide

Genbank accession no. NP_004346

Genbank version no. NP_004346.1 GI:10835071

Genbank record update date: Sep. 23, 2012 02:30 PM

CROSS-REFERENCES

Kudo, J., et al Nucleic Acids Res. 13 (24), 8827-8841 (1985)

Other Information

Official Symbol: CD74

Other Aliases: DHLAG, HLADG, II, la-GAMMA

Other Designations: CD74 antigen (invariant polypeptide of majorhistocompatibility complex, class II antigen-associated); HLA class IIhistocompatibility antigen gamma chain; HLA-DR antigens-associatedinvariant chain; HLA-DR-gamma; la-associated invariant chain; MHC HLA-DRgamma chain; gamma chain of class II antigens; p33

Antibodies

Immunomedics: hLL1 (Milatuzumab)—Berkova Z., et al Expert Opin InvestigDrugs. 2010 January; 19(1):141-9)

-   -   For example, see US20040115193 SEQ ID NOs: 19, 20, 21, 22, 23        and 24

Genmab: HuMax-CD74 (see website)

(75) Claudins—CLs (Claudins)

CROSS-REFERENCES

Offner S., et al Cancer Immunol Immunother. 2005 May; 54(5):431-45,Suzuki H., et al Ann NY Acad Sci. 2012 July; 1258:65-70)

In humans, 24 members of the family have been described—see literaturereference.

(76) EGFR (Epidermal Growth Factor Receptor)

Nucleotide

Genbank accession no. NM_005228

Genbank version no. NM_005228.3 GI:41927737

Genbank record update date: Sep. 30, 2012 01:47 PM

Polypeptide

Genbank accession no. NP_005219

Genbank version no. NP_005219.2 GI:29725609

Genbank record update date: Sep. 30, 2012 01:47 PM

CROSS-REFERENCES

Dhomen N S., et al Crit Rev Oncog. 2012; 17(1):31-50

Other Information

Official Symbol: EGFR

Other Aliases: ERBB, ERBB1, HER1, PIG61, mENA

Other Designations: avian erythroblastic leukemia viral (v-erb-b)oncogene homolog; cell growth inhibiting protein 40; cellproliferation-inducing protein 61; proto-oncogene c-ErbB-1; receptortyrosine-protein kinase erbB-1

Antibodies

BMS: Cetuximab (Erbitux)—Broadbridge V T., et al Expert Rev AnticancerTher. 2012 May; 12(5):555-65.

-   -   For example, see U.S. Pat. No. 6,217,866—ATTC deposit No. 9764.

Amgen: Panitumumab (Vectibix)—Argiles G., et al Future Oncol. 2012April; 8(4):373-89

-   -   For example, see U.S. Pat. No. 6,235,883 SEQ ID NOs: 23-38.

Genmab: Zalutumumab—Rivera F., et al Expert Opin Biol Ther. 2009 May;9(5):667-74.

YM Biosciences: Nimotuzumab—Ramakrishnan M S., et al MAbs. 2009January-February; 1(1):41-8.

-   -   For example, see U.S. Pat. No. 5,891,996 SEQ ID NOs: 27-34.

(77) Her3 (ErbB3)—ERBB3 (v-erb-b2 Erythroblastic Leukemia Viral OncogeneHomolog 3 (Avian))

Nucleotide

Genbank accession no. M34309

Genbank version no. M34309.1 GI:183990

Genbank record update date: Jun. 23, 2010 08:47 PM

Polypeptide

Genbank accession no. AAA35979

Genbank version no. AAA35979.1 GI:306841

Genbank record update date: Jun. 23, 2010 08:47 PM

CROSS-REFERENCES

Plowman, G. D., et al., Proc. Natl. Acad. Sci. U.S.A. 87 (13),4905-4909(1990)

Other Information

Official Symbol: ERBB3

Other Aliases: ErbB-3, HER3, LCCS2, MDA-BF-1, c-erbB-3, c-erbB3,erbB3-S, p180-ErbB3, p45-sErbB3, p85-sErbB3

Other Designations: proto-oncogene-like protein c-ErbB-3; receptortyrosine-protein kinase erbB-3; tyrosine kinase-type cell surfacereceptor HER3

Antibodies

Merimack Pharma: MM-121 (Schoeberl B., et al Cancer Res. 2010 Mar. 15;70(6):2485-2494)

-   -   For example, see US2011028129 SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7        and 8.

(78) RON—MST1R (Macrophage Stimulating 1 Receptor (c-Met-RelatedTyrosine Kinase))

Nucleotide

Genbank accession no. X70040

Genbank version no. X70040.1 GI:36109

Genbank record update date: Feb. 2, 2011 10:17 PM

Polypeptide

Genbank accession no. CCA49634

Genbank version no. CCA49634.1 GI:36110

Genbank record update date: Feb. 2, 2011 10:17 PM

CROSS-REFERENCES

Ronsin C., et al Oncogene 8 (5), 1195-1202 (1993)

Other Information

Official Symbol: MST1R

Other Aliases: CD136, CDw136, PTK8, RON

Other Designations: MSP receptor; MST1R variant RON30; MST1R variantRON62; PTK8 protein tyrosine kinase 8; RON variant E2E3; c-met-relatedtyrosine kinase; macrophage-stimulating protein receptor; p185-Ron;soluble RON variant 1; soluble RON variant 2; soluble RON variant 3;soluble RONvariant 4

(79) EPHA2 (EPH Receptor A2)

Nucleotide

Genbank accession no. BC037166

Genbank version no. BC037166.2 GI:33879863

Genbank record update date: Mar. 6, 2012 01:59 PM

Polypeptide

Genbank accession no. AAH37166

Genbank version no. AAH37166.1 GI:22713539

Genbank record update date: Mar. 6, 2012 01:59 PM

CROSS-REFERENCES

Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99 (26),16899-16903 (2002)

Other Information

Official Symbol: EPHA2

Other Aliases: ARCC2, CTPA, CTPP1, ECK

Other Designations: ephrin type-A receptor 2; epithelial cell receptorprotein tyrosine kinase; soluble EPHA2 variant 1; tyrosine-proteinkinase receptor ECK

Antibodies

Medimmune: 1C1 (Lee J W., et al Clin Cancer Res. 2010 May 1;16(9):2562-2570)

-   -   For example, see US20090304721A1 FIGS. 7 and 8.

(80) CD20—MS4A1 (Membrane-Spanning 4-Domains, Subfamily a, Member 1)

Nucleotide

Genbank accession no. M27394

Genbank version no. M27394.1 GI:179307

Genbank record update date: Nov. 30, 2009 11:16 AM

Polypeptide

Genbank accession no. AAA35581

Genbank version no. AAA35581.1 GI:179308

Genbank record update date: Nov. 30, 2009 11:16 AM

CROSS-REFERENCES

Tedder T. F., et al Proc. Natl. Acad. Sci. U.S.A. 85 (1), 208-212 (1988)

Other Information

Official Symbol: MS4A1

Other Aliases: B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7

Other Designations: B-lymphocyte antigen CD20; B-lymphocyte cell-surfaceantigen B1; CD20 antigen; CD20 receptor; leukocyte surface antigenLeu-16

Antibodies

Genentech/Roche: Rituximab—Abdulla N E., et al BioDrugs. 2012 Apr. 1;26(2):71-82.

-   -   For example, see U.S. Pat. No. 5,736,137, ATCC deposit No.        HB-69119.

GSK/Genmab: Ofatumumab—Nightingale G., et al Ann Pharmacother. 2011October; 45(10):1248-55.

-   -   For example, see US20090169550A1 SEQ ID NOs: 2, 4 and 5.

Immunomedics: Veltuzumab—Goldenberg D M., et al Leuk Lymphoma. 2010 May;51(5):747-55.

-   -   For example, see U.S. Pat. No. 7,919,273B2 SEQ ID NOs: 1, 2, 3,        4, 5 and 6.

(81) Tenascin C—TNC (Tenascin C)

Nucleotide

Genbank accession no. NM 002160

Genbank version no. NM_002160.3 GI:340745336

Genbank record update date: Sep. 23, 2012 02:33 PM

Polypeptide

Genbank accession no. NP_002151

Genbank version no. NP_002151.2 GI:153946395

Genbank record update date: Sep. 23, 2012 02:33 PM

CROSS-REFERENCES

Nies D. E., et al J. Biol. Chem. 266 (5), 2818-2823 (1991); Siri A., etal Nucleic Acids Res. 19 (3), 525-531 (1991)

Other Information

Official Symbol: TNC

Other Aliases: 150-225, GMEM, GP, HXB, JI, TN, TN-C

Other Designations: GP 150-225; cytotactin;glioma-associated-extracellular matrix antigen; hexabrachion (tenascin);myotendinous antigen; neuronectin; tenascin; tenascin-C isoform14/AD1/16

Antibodies

Philogen: G11 (von Lukowicz T., et al J Nucl Med. 2007 April;48(4):582-7) and F16 (Pedretti M., et al Lung Cancer. 2009 April;64(1):28-33)

-   -   For example, see U.S. Pat. No. 7,968,685 SEQ ID NOs: 29, 35, 45        and 47.

(82) FAP (Fibroblast Activation Protein, Alpha)

Nucleotide

Genbank accession no. U09278

Genbank version no. U09278.1 GI:1888315

Genbank record update date: Jun. 23, 2010 09:22 AM

Polypeptide

Genbank accession no. AAB49652

Genbank version no. AAB49652.1 GI:1888316

Genbank record update date: Jun. 23, 2010 09:22 AM

CROSS-REFERENCES

Scanlan, M. J., et al Proc. Natl. Acad. Sci. U.S.A. 91 (12), 5657-5661(1994)

Other Information

Official Symbol: FAP

Other Aliases: DPPIV, FAPA

Other Designations: 170 kDa melanoma membrane-bound gelatinase; integralmembrane serine protease; seprase

(83) DKK-1 (Dickkopf 1 Homolog (Xenopus laevis)

Nucleotide

Genbank accession no. NM_012242

Genbank version no. NM_012242.2 GI:61676924

Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_036374

Genbank version no. NP_036374.1 GI:7110719

Genbank record update date: Sep. 30, 2012 01:48 PM

CROSS-REFERENCES

Fedi P. et al J. Biol. Chem. 274 (27), 19465-19472 (1999)

Other Information

Official Symbol: DKK1

Other Aliases: UNQ492/PRO1008, DKK-1, SK

Other Designations: dickkopf related protein-1; dickkopf-1 like;dickkopf-like protein 1; dickkopf-related protein 1; hDkk-1

Antibodies

Novartis: BHQ880 (Fulciniti M., et al Blood. 2009 Jul. 9;114(2):371-379)

-   -   For example, see US20120052070A1 SEQ ID NOs: 100 and 108.

(84) CD52 (CD52 Molecule)

Nucleotide

Genbank accession no. NM_001803

Genbank version no. NM 001803.2 GI:68342029

Genbank record update date: Sep. 30, 2012 01:48 PM

Polypeptide

Genbank accession no. NP_001794

Genbank version no. NP_001794.2 GI:68342030

Genbank record update date: Sep. 30, 2012 01:48 PM

CROSS-REFERENCES

Xia M. Q., et al Eur. J. Immunol. 21 (7),1677-1684 (1991)

Other Information

Official Symbol: CD52

Other Aliases: CDW52

Other Designations: CAMPATH-1 antigen; CD52 antigen (CAMPATH-1 antigen);CDW52 antigen (CAMPATH-1 antigen); cambridge pathology 1 antigen;epididymal secretory protein E5; he5; human epididymis-specific protein5

Antibodies

Alemtuzumab (Campath)—Skoetz N., et al Cochrane Database Syst Rev. 2012Feb. 15; 2:CD008078.

-   -   For example, see Drugbank Acc. No. DB00087 (BIOD00109, BTDO0109)

(85) CS1—SLAMF7 (SLAM Family Member 7)

Nucleotide

Genbank accession no. NM_021181

Genbank version no. NM_021181.3 GI:1993571

Genbank record update date: Jun. 29, 2012 11:24 AM

Polypeptide

Genbank accession no. NP_067004

Genbank version no. NP_067004.3 GI:19923572

Genbank record update date: Jun. 29, 2012 11:24 AM

CROSS-REFERENCES

Boles K. S., et al Immunogenetics 52 (3-4), 302-307 (2001)

Other Information

Official Symbol: SLAMF7

Other Aliases: UNQ576/PRO1138, 19A, CD319, CRACC, CS1

Other Designations: 19A24 protein; CD2 subset 1; CD2-like receptoractivating cytotoxic cells; CD2-like receptor-activating cytotoxiccells; membrane protein FOAP-12; novel LY9 (lymphocyte antigen 9) likeprotein; protein 19A

Antibodies

BMS: elotuzumab/HuLuc63 (Benson D M., et al J Clin Oncol. 2012 Jun. 1;30(16):2013-2015)

-   -   For example, see US20110206701 SEQ ID NOs: 9, 10, 11, 12, 13,        14, 15 and 16.

(86) Endoglin—ENG (Endoglin)

Nucleotide

Genbank accession no. AF035753

Genbank version no. AF035753.1 GI:3452260

Genbank record update date: Mar. 10, 2010 06:36 PM

Polypeptide

Genbank accession no. AAC32802

Genbank version no. AAC32802.1 GI:3452261

Genbank record update date: Mar. 10, 2010 06:36 PM

CROSS-REFERENCES

Rius C., et al Blood 92 (12), 4677-4690 (1998)

Official Symbol: ENG

Other Information

Other Aliases: RP11-228B15.2, CD105, END, HHT1, ORW, ORW1

Other Designations: CD105 antigen

(87) Annexin A1—ANXA1 (Annexin A1)

Nucleotide

Genbank accession no. X05908

Genbank version no. X05908.1 GI:34387

Genbank record update date: Feb. 2, 2011 10:02 AM

Polypeptide

Genbank accession no. CCA29338

Genbank version no. CCA29338.1 GI:34388

Genbank record update date: Feb. 2, 2011 10:02 AM

CROSS-REFERENCES

Wallner B. P., et al Nature 320 (6057), 77-81 (1986)

Other Information

Official Symbol: ANXA1

Other Aliases: RP11-71A24.1, ANX1, LPC1

Other Designations: annexin I (lipocortin I); annexin-1; calpactin 1l;calpactin-2; chromobindin-9; lipocortin I; p35; phospholipase A2inhibitory protein

(88) V-CAM (CD106)—VCAM1 (Vascular Cell Adhesion Molecule 1)

Nucleotide

Genbank accession no. M60335

Genbank version no. M60335.1 GI:340193

Genbank record update date: Jun. 23, 2010 08:56 AM

Polypeptide

Genbank accession no. AAA61269

Genbank version no. AAA61269.1 GI:340194

Genbank record update date: Jun. 23, 2010 08:56 AM

CROSS-REFERENCES

Hession C., et al J. Biol. Chem. 266 (11), 6682-6685 (1991)

Other Information

Official Symbol VCAM1

Other Aliases: CD106, INCAM-100

Other Designations: CD106 antigen; vascular cell adhesion protein 1

Antibody Sequences

Anti-Integrin avB6

RHAB6.2 QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTAVPNLRGDLQVLAQKVAGPYPFDYWGQGTLVTVSS RHCB6.2QVQLVQSGAEVKKPGASVKVSCKASGYTFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTITTDTSASTAYMELSSLRSEDTAVYYCARGTPTAVPNLRGDLQVLAQKVAGPYPFDYWGQGTLVTVSS RHFQVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGT PTGPYYFDYWGQGTLVTVSSRHFB6 QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDTEYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTAVPNLRGDLQVLAQKVAGPYYFDYWGQGTLVTVSS RHAY100bPQVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTEYAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGT PTGPYPFDYWGQGTLVTVSSRKF ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGG TKVEIK RKFL36L50ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWLQQKPGQAPRLLIYLTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGG TKVEIK RKCEIVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGG TKVEIK

Anti-CD33

CD33 Hum195 VH QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGR PAMDYWGQGTLVTVSSCD33 Hum195 VK DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPW TFGQGTKVEIK

Anti-CD19

CD19 B4 resurfaced VH QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGS NPYYYAMDYWGQGTSVTVSSCD19 B4 resurfaced VK EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTK LEIK

Anti-Her2

Herceptin VH chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSHerceptin VL chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK

Anti-CD25

Simulect VK (also known as Basiliximab)QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYTFGGGTK LEIK Simulect VHQLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQRPGQGLEWIGAIYPGNSDTSYNQKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSRDYGY YFDFWGQGTTLTVSS

Anti-PSMA

Deimmunised VH ′1EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTYNQKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSSDeimmunised VK ′1DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPSRFSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK Deimmunised VH1 ′5EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTGVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH2 ′5EVKLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH3 ′5EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VH4 ′5EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFATHYAESVKGRFTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSSDeimmunised VK1 ′5NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFTGSGSATDFTLTISSLQTEDLADYYCGQSYTFPYTFGQGTKLEMK Deimmunised VK2 ′5NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK3 ′5NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK4 ′5NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPDRFSGSGSGTDFTLTISSLQAEDEADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK DI 5NIVMTQFPKSMSASAGERMTLTCKASENVGTYVSWYQQKPTQSPKMLIYGASNRFTGVPDRFSGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFGGGTKLEMK Deimmunised VH DI ′5EVKLEESGGGLVQPGGSMKISCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKSSVYLQMNSLRAEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHA ′5EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFATHYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHB ′5EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHC ′5EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHD ′5EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFATHYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHE ′5EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRFTISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHF ′5EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RHG ′5EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFATHYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSSHumanised RKA ′5DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKB ′5DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKC ′5DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKD ′5DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKE ′5NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPDRFTGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKF ′5NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSRFSGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKG ′5NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPDRFTGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

The parent antibody may also be a fusion protein comprising analbumin-binding peptide (ABP) sequence (Dennis et al. (2002) “AlbuminBinding As A General Strategy For Improving The Pharmacokinetics OfProteins” J Biol Chem. 277:35035-35043; WO 01/45746). Antibodies of theinvention include fusion proteins with ABP sequences taught by: (i)Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV,page 35038; (ii) US 2004/0001827 at [0076]; and (iii) WO 01/45746 atpages 12-13, and all of which are incorporated herein by reference.

In one embodiment, the antibody has been raised to target specific thetumour related antigen α_(v)β₆.

The cell binding agent may be labelled, for example to aid detection orpurification of the agent either prior to incorporation as a conjugate,or as part of the conjugate. The label may be a biotin label. In anotherembodiment, the cell binding agent may be labelled with a radioisotope.

Connection of Linker Unit to Ligand Unit

The Ligand unit is connected to the Linker unit through a disulfidebond.

In one embodiment, the connection between the Ligand unit and the DrugLinker is formed between a thiol group of a cysteine residue of theLigand unit and a maleimide group of the Drug Linker unit.

The cysteine residues of the Ligand unit may be available for reactionwith the functional group of the Linker unit to form a connection. Inother embodiments, for example where the Ligand unit is an antibody, thethiol groups of the antibody may participate in interchain disulfidebonds. These interchain bonds may be converted to free thiol groups bye.g. treatment of the antibody with DTT prior to reaction with thefunctional group of the Linker unit.

In some embodiments, the cysteine residue is an introduced into theheavy or light chain of an antibody. Positions for cysteine insertion bysubstitution in antibody heavy or light chains include those describedin Published U.S. Application No. 2007-0092940 and International PatentPublication WO2008070593, which are incorporated herein.

Methods of Treatment

The compounds of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a conjugate of formula. The term“therapeutically effective amount” is an amount sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

A conjugate may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Examples of treatments and therapies include,but are not limited to, chemotherapy (the administration of activeagents, including, e.g. drugs; surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a conjugate of formula I, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The Conjugates can be used to treat proliferative disease and autoimmunedisease. The term “proliferative disease” pertains to an unwanted oruncontrolled cellular proliferation of excessive or abnormal cells whichis undesired, such as, neoplastic or hyperplastic growth, whether invitro or in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Other cancers of interest include, but are not limitedto, haematological; malignancies such as leukemias and lymphomas, suchas non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone,mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers ofB or T cell origin.

Examples of autoimmune disease include the following: rheumatoidarthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis,allergic encephalomyelitis), psoriatic arthritis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Graves' disease, glomerulonephritis, autoimmune hepatologicaldisorder, inflammatory bowel disease (e.g., Crohn's disease),anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetesmellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of Blymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g.,rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments,the autoimmune disorder is a T cell-mediated immunological disorder.

In some embodiments, the amount of the Conjugate administered rangesfrom about 0.01 to about 10 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.01 to about 5mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.05 to about 5 mg/kg per dose. In someembodiments, the amount of the Conjugate administered ranges from about0.1 to about 5 mg/kg per dose. In some embodiments, the amount of theConjugate administered ranges from about 0.1 to about 4 mg/kg per dose.In some embodiments, the amount of the Conjugate administered rangesfrom about 0.05 to about 3 mg/kg per dose. In some embodiments, theamount of the Conjugate administered ranges from about 0.1 to about 3mg/kg per dose. In some embodiments, the amount of the Conjugateadministered ranges from about 0.1 to about 2 mg/kg per dose.

Drug Loading

The drug loading (p) is the average number of PBD drugs per cell bindingagent, e.g. antibody. Where the compounds of the invention are bound tocysteines, drug loading may range from 1 to 8 drugs (D) per cell bindingagent, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties arecovalently attached to the cell binding agent. Compositions ofconjugates include collections of cell binding agents, e.g. antibodies,conjugated with a range of drugs, from 1 to 8. Where the compounds ofthe invention are bound to lysines, drug loading may range from 1 to 80drugs (D) per cell binding agent, although an upper limit of 40, 20, 10or 8 may be preferred. Compositions of conjugates include collections ofcell binding agents, e.g. antibodies, conjugated with a range of drugs,from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, andelectrophoresis. The quantitative distribution of ADC in terms of p mayalso be determined. By ELISA, the averaged value of p in a particularpreparation of ADC may be determined (Hamblett et al (2004) Clin. CancerRes. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852).However, the distribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis. Suchtechniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading, e.g. p>5, may cause aggregation,insolubility, toxicity, or loss of cellular permeability of certainantibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with theDrug Linker (A or B). Only the most reactive lysine groups may reactwith an amine-reactive linker reagent. Also, only the most reactivecysteine thiol groups may react with a thiol-reactive linker reagent.Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which may be linked to a drug moiety. Mostcysteine thiol residues in the antibodies of the compounds exist asdisulfide bridges and must be reduced with a reducing agent such asdithiothreitol (DTT) or TCEP, under partial or total reducingconditions. The loading (drug/antibody ratio) of an ADC may becontrolled in several different manners, including: (i) limiting themolar excess of Drug Linker (A or B) relative to antibody, (ii) limitingthe conjugation reaction time or temperature, and (iii) partial orlimiting reductive conditions for cysteine thiol modification.

Certain antibodies have reducible interchain disulfides, i.e. cysteinebridges. Antibodies may be made reactive for conjugation with linkerreagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by engineering one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541teaches engineering antibodies by introduction of reactive cysteineamino acids.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al(2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485;WO2009/052249). The engineered cysteine thiols may react with linkerreagents or the drug-linker reagents of the present invention which havethiol-reactive, electrophilic groups such as maleimide or alpha-haloamides to form ADC with cysteine engineered antibodies and the PBD drugmoieties. The location of the drug moiety can thus be designed,controlled, and known. The drug loading can be controlled since theengineered cysteine thiol groups typically react with thiol-reactivelinker reagents or drug-linker reagents in high yield. Engineering anIgG antibody to introduce a cysteine amino acid by substitution at asingle site on the heavy or light chain gives two new cysteines on thesymmetrical antibody. A drug loading near 2 can be achieved with nearhomogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

Thus the antibody-drug conjugate compositions of the invention includemixtures of antibody-drug conjugate compounds where the antibody has oneor more PBD drug moieties and where the drug moieties may be attached tothe antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepinegroups per cell binding agent is in the range 1 to 20. In someembodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4,and 4 to 8.

In some embodiments, there is one dimer pyrrolobenzodiazepine group percell binding agent.

General Synthetic Routes

The synthesis of PBD compounds is extensively discussed in the followingreferences, which discussions are incorporated herein by reference:

a) WO 00/12508 (pages 14 to 30);

b) WO 2005/023814 (pages 3 to 10);

c) WO 2004/043963 (pages 28 to 29); and

d) WO 2005/085251 (pages 30 to 39).

Synthesis Route

The Drug Linker compounds of the present invention (A and B) may besynthesised according to the Examples.

Synthesis of Drug Conjugates

Conjugates can be prepared as previously described. Antibodies can beconjugated to the Drug Linker compounds (A or B) as described inDoronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly,antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37°C. The progress of the reaction, which reduces interchain disulfides, ismonitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) andallowed to proceed until the desired level of thiols/mAb is achieved.The reduced antibody is then cooled to 0° C. and alkylated with 1.5equivalents of maleimide drug-linker per antibody thiol. After 1 hour,the reaction is quenched by the addition of 5 equivalents of N-acetylcysteine. Quenched drug-linker is removed by gel filtration over a PD-10column. The ADC is then sterile-filtered through a 0.22 μm syringefilter. Protein concentration can be determined by spectral analysis at280 nm and 329 nm, respectively, with correction for the contribution ofdrug absorbance at 280 nm. Size exclusion chromatography can be used todetermine the extent of antibody aggregation, and RP-HPLC can be used todetermine the levels of remaining NAC-quenched drug-linker.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In some embodiments, the C11 substituent may be in the followingstereochemical arrangement relative to neighbouring groups:

In other embodiments, the C11 substituent may be in the followingstereochemical arrangement relative to neighbouring groups:

In one embodiment of the present invention, the compound of formula IIIis A.

In one embodiment of the present invention, the compound of formula IIIis B.

In one embodiment of the present invention, the Drug Linker unit offormula III is D^(L)-A.

In one embodiment of the present invention, the Drug Linker unit offormula III is D^(L)-B.

EXAMPLES

Reaction progress was monitored by thin-layer chromatography (TLC) usingMerck Kieselgel 60 F254 silica gel, with fluorescent indicator onaluminium plates. Visualisation of TLC was achieved with UV light oriodine vapour unless otherwise stated. Flash chromatography wasperformed using Merck Kieselgel 60 F254 silica gel. Extraction andchromatography solvents were bought and used without furtherpurification from VWR, U.K. All chemicals were purchased from Aldrich.

Proton NMR chemical shift values were measured on the delta scale at 400MHz using a Bruker AV400. The following abbreviations have been used: s,singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m,multiplet; br, broad. Coupling constants are reported in Hz. Columnchromatography was performed on an Isolera (Biotage) automated systemusing normal phase SNAP cartridges.

The LC/MS Conditions were as Follow:

LCMS data were obtained using a Shimadzu Nexera series LC/MS with aShimadzu LCMS-2020 quadrupole MS, with Electrospray ionisation. Mobilephase A—0.1% formic acid in water. Mobile phase B—0.1% formic acid inacetonitrile.

Short run gradient: initial composition was 5% B held over 0.25 min,then increase from 5% B to 100% B over a 2 min period. The compositionwas held for 0.50 min at 100% B, then returned to 5% B in 0.05 minutesand hold there for 0.05 min. Total gradient run time equals 3 min. Flowrate 0.8 mL/min. Wavelength detection range: 190 to 800 nm. Oventemperature: 50° C. Column: Waters Acquity UPLC BEH Shield RP18 1.7 μm2.1×50 mm.

Long run gradient: initial composition 5% B held over 1 min, thenincrease from 5% B to 100% B over a 9 min period. The composition washeld for 2 min at 100% B, then returned to 5% B in 0.10 minutes and holdthere for 3 min. Total gradient run time equals 15 min. Flow rate 0.6mL/min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50°C. Column: ACE Excel 2 C18-AR, 2μ, 3.0×100 mm.

Example 1 (a) (S)-2-(methoxycarbonyl)-4-methylenepyrrolidinium Chloride(3)

Commercially available proline derivative (1) was obtained fromOmegachem

(i) (S)-1-tert-butyl 2-methyl 4-methylenepyrrolidine-1,2-dicarboxylate(2)

Potassium carbonate (19.92 g, 14 mmol, 3.0 eq.) was added to a stirredsolution of the carboxylic acid 1 (10.92 g, 48 mmol, 1.0 eq.) in DMF(270 mL). The resulting white suspension was stirred at room temperaturefor 30 mins, at which point iodomethane (21.48 g, 9.5 mL, 151 mmol, 3.15eq.) was added. The reaction mixture was allowed to stir at roomtemperature for 3 days. The DMF was removed by rotary evaporation underreduced pressure to afford a yellow residue which was partitionedbetween ethylacetate and water. The organic layer was separated and theaqueous phase was extracted with ethylacetate. The combined organiclayers were washed with water brined and dried over magnesium sulphate.The ethylacetate was removed by rotary evaporation under reducedpressure to give the crude product as a yellow oil. The crude productwas purified by flash chromatography [85% n-hexane/15% ethylacetate] toafford the product as a colourless oil (10.74 g, 93%).

(ii) (S)-2-(methoxycarbonyl)-4-methylenepyrrolidinium Chloride (3)

A solution of 4 M hydrochloric acid in dioxane (63 mL, 254.4 mmol, 4.5eq.) was added to the Boc protected C-ring fragment 2 (13.67 g, 56.6mmol, 1.0 eq.) at room temperature. Effervescence was observedindicating liberation of CO₂ and removal of the Boc group. The productprecipitated as a white solid and additional dioxane was added tofacilitate stirring the reaction mixture was allowed to stir for an hourand then diluted with diethyl ether. The precipitated product wascollected by vacuum filtration and washed with additional diethyl ether.Air drying afforded the desired product as a white powder (9.42 g, 94%).

(b) Tert-Butyl(5-(3-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(12)

(i) 1′,3′-Bis[2-methoxy-4-(methoxycarbonyl)phenoxy]propane (5)

Diisopropyl azodicarboxylate (71.3 mL, 73.2 g, 362 mmol) was addeddrop-wise over a period of 60 min to an overhead stirred solution ofmethyl vanillate 4 (60 g, 329 mmol) and Ph₃P (129.4 g, 494 mmol) inanhydrous THE (800 mL) at 0-5° C. (ice/acetone) under a nitrogenatmosphere. The reaction mixture was allowed to stir at 0-5° C. for anadditional 1 h after which time a solution of 1,3-propanediol (11.4 mL,12.0 g, 158 mmol) in THE (12 mL) was added drop-wise over a period of 20min. The reaction mixture was allowed to warm to room temperature andstirred for 5 days. The resulting white precipitate 3 was collected byvacuum filtration, washed with THE and dried in a vacuum desiccator toconstant weight. Yield=54.68 g (84% based on 1,3-propanediol).Analytical Data: Purity satisfactory by LC/MS 3.20 min (ES+) m/z(relative intensity) 427 ([M+Na]^(+⋅), 10); ¹H NMR (400 MHz, CDCl₃) δ67.64 (dd, 2H, J=1.8, 8.3 Hz), 7.54 (d, 2H, J=1.8 Hz), 6.93 (d, 2H,J=8.5 Hz), 4.30 (t, 4H, J=6.1 Hz), 3.90 (s, 6H), 3.89 (s, 6H), 2.40 (p,2H, J=6.0 Hz).

(ii) 1′,3′-Bis[2-methoxy-4-(methoxycarbonyl)-5-nitrophenoxy]propane (6)

Solid Cu(NO₃)₂.3H₂O (81.54 g, 337.5 mmol) was added slowly to anoverhead stirred slurry of the bis-ester 5 (54.68 g, 135 mmol) in aceticanhydride (650 mL) at 0-5° C. (ice/acetone). The reaction mixture wasallowed to stir for 1 h at 0-5° C. and then allowed to warm to roomtemperature. A mild exotherm (c. 40-50° C.), accompanied by thickeningof the mixture and evolution of NO₂ was observed at this stage.Additional acetic anhydride (300 mL) was added and the reaction mixturewas allowed to stir for 16 h at room temperature. The reaction mixturewas poured onto ice (˜1.5 L), stirred and allowed to return to roomtemperature. The resulting yellow precipitate was collected by vacuumfiltration and dried in a desiccator to afford the desired bis-nitrocompound 6 as a yellow solid. Yield=66.7 g (100%). Analytical Data:Purity satisfactory by LC/MS 3.25 min (ES+) m/z (relative intensity) 517([M+Na]^(+⋅), 40); ¹H NMR (400 MHz, CDCl₃) δ 7.49 (s, 2H), 7.06 (s, 2H),4.32 (t, 4H, J=6.0 Hz), 3.95 (s, 6H), 3.90 (s, 6H), 2.45-2.40 (m, 2H).See ref Thurston 1996.

(iii) 1′3′-Bis(4-carboxy-2-methoxy-5-nitrophenoxy) propane (7)

A slurry of the methyl ester 6 (66.7 g, 135 mmol) in THE (700 mL) wastreated with 1N NaOH (700 mL) and the reaction mixture was allowed tostir vigorously at room temperature. After 4 days stirring, the slurrybecame a dark coloured solution which was subjected to rotaryevaporation under reduced pressure to remove THF. The resulting aqueousresidue was acidified to pH 1 with concentrated HCl and the colourlessprecipitate 7 was collected and dried thoroughly in a vacuum oven (50°C.). Yield=54.5 g (87%). Analytical Data: Purity satisfactory by LC/MS2.65 min (ES+) m/z (relative intensity) 489 ([M+Na]^(+⋅), 30); ¹H NMR(400 MHz, DMSO-d₆) 7.62 (s, 2H), 7.30 (s, 2H), 4.29 (t, 4H, J=6.0 Hz),3.85 (s, 6H), 2.30-2.26 (m, 2H).

(iv) Dimethyl1,1′-(4,4′-(propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitrobenzoyl))(2S,2'S)-bis(4-methylenepyrrolidine-2-carboxylate)(8)

A catalytic amount of anhydrous DMF (2.4 mL) was added to a stirredsuspension of oxalyl chloride (14.7 g, 9.8 mL, 115.8 mmol, 3 eq.) anddimer core 7 (18 g, 38.6 mmol, 1 eq.) in anhydrous DCM (500 mL) at roomtemperature. Vigorous effervescence was observed after the addition ofDMF and the reaction mixture was allowed to stir for 18 h in a roundbottom flask fitted with a calcium chloride drying tube. The resultingclear solution was evaporated under reduced pressure and the solidtriturated with ether. The solid product was collected by vacuumfiltration, washed with additional ether and dried in vacuo at 40° C.for 1.5 h. This solid was then added portion wise to a suspension of theC-ring 3 (15.1 g, 84.9 mmol, 2.2 eq.) and TEA (19.5 g, 27 ml, 119.6mmol, 5 eq.) in dry DCM (375 mL), maintaining the temperature between−40 and −50° C. with the aid of a dry ice/acetonitrile bath. Thereaction mixture was allowed to stir at −40° C. for 1h and then allowedto warm to room temperature at which point LCMS indicated the completeconsumption of the starting material. The reaction mixture was dilutedwith additional DCM and washed sequentially with aqueous hydrochloricacid (1M, 2×200 mL), saturated aqueous sodium bicarbonate (2×250 mL),water (250 mL), brine (250 mL), dried (MgSO₄). DCM was removed by rotaryevaporation under reduced pressure to afford the product as a yellowfoam (25.72 g, 94%). Analytical Data: RT 1.59 min; MS (ES⁺) m/z(relative intensity) 713 ([M+H]^(+⋅), 100)

(v)((Propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone)(9)

Solid lithium borohydride (3.18 g, 146 mmol, 3 eq.) was added in oneportion to a solution of the ester 8 (34.72 g, 48.7 mmol, 1 eq.) in dryTHE (350 mL) under a nitrogen atmosphere at 0° C. (ice bath). Thereaction mixture was allowed to stir at 0° C. for 30 mins and thenallowed to warm to room temperature at which point precipitation of anorange gum was observed. The reaction mixture was allowed to stir atroom temperature for a further 2 hours and then cooled in an ice bathand treated with water to give a yellow suspension. Hydrochloric acid(1M) was carefully added until effervescence ceased. The reactionmixture was extracted with ethylacetate (×4) and the combined organiclayers were washed with water (×1), brine (×1) and dried (MgSO₄).Ethylacetate was removed by rotary evaporation under reduced pressure togive a yellow foam. Purification by flash column chromatography[gradient elution DCM/MeOH 0% to 5% in 1% increments] gave the productas a pale yellow foam (23.1 g, 72%). Analytical Data: RT 1.23 min; MS(ES⁺) m/z (relative intensity) 657 ([M+H]^(+⋅), 100)

(vi)((Propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-1-yl)methanone)(10)

A solution of the bis-alcohol 9 (10 g, 15.2 mmol, 1 eq.),t-butyldimethylsilylchloride (5.97 g, 39.6 mmol, 2.6 eq.) and imidazole(5.38 g, 79 mmol, 5.2 eq.) in dry DMF (80 ml) was stirred at roomtemperature for 3h. The reaction mixture was poured into water (500 mL)to give a yellow precipitate. The mixture was extracted with DCM (4×100mL) and the combined extracts were washed with water and brine, dried(MgSO₄) and evaporated under reduced pressure to give a viscous yellowoil. Purification by column chromatography [biotage isolera, gradientelution hexane 60%/EtOAc 40% to EtOAc 100%, 8 column volumes 100 g snapultra@ cartridge] gave the product as a yellow foam (11.8 g, 88%).Analytical Data: RT 2.20 min; MS (ES⁺) m/z (relative intensity) 885([M+H]⁺, 100), 907 ([M+Na]^(+⋅), 50)

(vii)((Propane-1,3-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-1-yl)methanone)(11)

Zinc powder (31.9 g, 488 mmol, 40 eq.) was activated bystirring/sonication with 1M HCl for 10 min. The Zinc was filteredwashing with 1M HCl, water (×3) and MeOH (×2). The activated Zinc wasadded to a solution of the nitro-TBS compound 10 (10.8 g, 12.2 mmol, 1eq.) in MeOH (88 mL) and 5% formic acid/MeOH solution (440 mL). Thetemperature rose to 37° C. and the reaction mixture changed from ayellow to a colourless solution. Once the exotherm had subsided (20min.) the reaction was shown to be complete by LCMS. The reactionmixture was filtered through celite washing with EtOAc. The EtOAcportion was washed with saturated bicarbonate solution (×4) [cautioneffervescence!], water (×1), brine (×1), dried (MgSO4) and evaporatedunder reduced pressure to give a yellow solid. Purification by flashcolumn chromatography [n-hexane/EtOAc 50/50 v/v to EtOAc 100% in 10%increments] gave the product as a yellow foam (9.5 g, 86%). AnalyticalData: RT 2.12 min; MS (ES⁺) m/z (relative intensity) 825 ([M+H]^(+⋅),60), 847 ([M+Na]^(+⋅), 30)

(viii) tert-Butyl(5-(3-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(12)

A solution of the bis-aniline 11 (3.27 g, 3.96 mmol) anddi-t-butyldicarbonate (0.85 g, 3.96 mmol) in dry THE (125 mL) wereheated under reflux for 24h. The reaction mixture was cooled and thesolvent evaporated under reduced pressure. The residue was purified byflash column chromatography [n-hexane/EtOAc 50/50 v/v to EtOAc 100% in10% increments then EtOAc/MeOH 98/2 v/v] to give the desired product asa yellow foam (1.63 g, 44%). Analytical Data: RT 2.28 min; MS (ES⁺) m/z(relative intensity) 925 ([M+H]^(+⋅), 70), 947 ([M+Na]^(+⋅), 100)

(c) Alloc-Val-Ala-PABOH (17)

(i) Alloc-Val-OH (14)

Allyl chloroformate (41 g, 36.2 mL, 0.34 mol, 1.2 eq.) was addeddropwise to a stirred solution of L-valine 13 (33.25 g, 0.28 mol, 1 eq.)and potassium carbonate (58.9 g, 0.426 mol, 1.5 eq.) in water (650 mL)and THE (650 mL). The reaction mixture was stirred at room temperaturefor 18h. The THE was evaporated under reduced pressure and the remainingsolution was extracted with diethyl ether (or MTBE) (×2). The aqueousportion was acidified to pH 2 with conc. HCl and extracted with DCM(×3). The combined organic extracts were washed with brine (×1), dried(MgSO₄) and evaporated under reduced pressure to give a colourless oil(57.1 g). This was used in the next step without further purification.

(ii) Alloc-Val-OSu (15)

To a stirred solution of compound 14 (57.1 g, 0.28 mol, 1 eq.) andN-hydroxysuccinimide (32.68 g, 0.28 mol, 1 eq.) in dry THE (800 mL) wasadded dicyclohexylcarbodiimide (58.6 g, 0.28 mol, 1 eq.). The reactionmixture was stirred at room temperature for 18h. The reaction mixturewas filtered. The solid was washed with THE and the combined filtratewas concentrated under reduced pressure. The oil/solid residue wasre-dissolved in DCM and left to stand at 0° C. for 30 min. Thesuspension was filtered washing with cold DCM. Evaporation of thefiltrate under reduced pressure gave the succinimide ester as a whitesolid which was used in the next step without further purification.

(iii) Alloc-Val-Ala-OH (16)

A solution of Alloc-Val-OSu 15 (11.67 g, 39.0 mmol, 1 eq.) in THE (50mL) was added to a solution of H-Ala-OH (3.66 g, 41.08 mmoL, 1.05 eq.)and NaHCO₃ (3.61 g, 43.03 mmol, 1.1 eq.) in THE (100 mL) and H₂O (100mL). The mixture was stirred at room temperature for 72h and the THE wasevaporated under reduced pressure. The pH was adjusted to 3-4 withcitric acid to precipitate a white gum. This was extracted withethylacetate (6×150 mL) and the combined extracts were washed with H₂O(200 mL), brine (200 mL), dried (MgSO₄) and evaporated under reducedpressure to give a white solid. Trituration with diethyl ether (xs)afforded the pure product as a white powder (7.93 g, 74%). AnalyticalData: RT 2.17 min; MS (ES⁺) m/z (relative intensity) 295 ([M+Na]^(+⋅),63), 273 ([M+1]^(+⋅,) 60).

(iv) Alloc-Val-Ala-PABOH (17)

EEDQ (4.79 g, 19.3 mmol, 1.05 eq.) was added to a solution ofp-aminobenzyl alcohol (2.38 g, 19.3 mmol, 1.05 eq.) and Alloc-Val-Ala-OH16 (5.02 g, 18.4 mmol, 1.0 eq) in dry THE (100 mL). The mixture wasstirred at room temperature for 72h. The solvent was evaporated underreduced pressure to give a pale brown solid. The solid was trituratedwith diethyl ether and filtered washing with an excess of diethyl ether.This afforded the product as a white solid (6.2 g, 89%). AnalyticalData: RT 2.50 min; MS (ES⁺) m/z (relative intensity) 400.6 ([M+Na]^(+⋅).50), 378.6 ([M+1]^(+⋅), 60).

(d)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(23)

(i)tert-butyl(5-(3-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(18)

Triethylamine (0.38 g, 0.53 mL, 3.8 mmol, 2.2 eq.) was added to astirred solution of the mono-boc protected bis-aniline (12) (1.6 g, 1.72mmol, 1.0 eq.) and triphosgene (0.184 g, 0.62 mmol, 0.36 eq.) in dry THE(25 mL) under a nitrogen atmosphere at room temperature. The reactionmixture was heated to 40° C., after 5 min a sample was treated withmethanol and analysed by LCMS as the methyl carbamate. Analytical Data:RT 2.32 min; MS (ES⁺) m/z (relative intensity) 983 ([M+H]^(+⋅), 55),1005 ([M+Na]^(+⋅), 100) A solution/suspension of the benzyl-alcohol (17)(1.52 g, 2.35 mmol, 1.4 eq.) and triethylamine (0.26 g, 0.36 mL 2.6mmol, 1.5 eq.) in dry THE (40 mL) was run in from a dropping funnel tothe freshly prepared isocyanate. The reaction mixture was stirred at 40°C. for 2.5h. The reaction mixture was allowed to cool, filtered and thefiltrate evaporated to dryness to afford the crude product as a yellowoil which was purified by flash column chromatography [n-hexane/EtOAc50/50 v/v] which gave the product as a yellow glass (1.192 g). The mixedfractions were purified by flash column chromatography [CHCl₃/MeOH 0% to1%] to give a further amount of product (0.22 g). The material wascombined to give the product as a yellow foam (1.41 g, 63%). AnalyticalData: RT 2.27 min; MS (ES⁺) m/z (relative intensity) 1328 ([M+H]^(+⋅),30), 1350 ([M+Na]^(+⋅), 100)

(ii) tert-butyl(5-(3-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(19)

A 1.0M solution of TBAF in THE (2.34 mL, 2.34 mmol, 2.2 eq.) was addedto a solution of the bis-TBS compound (18) (1.41 g, 1.06 mmol, 1.0 eq.)in anhydrous THE (12 mL). The mixture was stirred at room temperaturefor 30 min., the solvent was removed under reduced pressure and theresidue purified by flash column chromatography [CHCl₃/MeOH 0% to 4% in1% increments] to give the desired product as a white foam (0.98 g,84%). Analytical Data: RT 1.62 min; MS (ES⁺) m/z (relative intensity)1100 ([M+H]^(+⋅), 60), 1122 ([M+Na]^(+⋅), 100)

(iii)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-y)oxy)propoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(20)

IBX (45 wt %, 1.3 g, 2.09 mmol, 2.4 eq.) was added to a solution of thebis-alcohol 19 (0.959 g, 0.87 mmol, 1.0 eq.) in anhydrous DMSO (25 mL).The solution was stirred at 30° C. for 18h. LCMS analysis indicated thepresence of a small amount of partially cyclised material. A furtherportion of IBX (45 wt %, 0.049 g, 0.17 mmol, 0.2 eq.) was added and thereaction was continued for a further 18h. The reaction mixture waspoured into water (200 mL) and the resultant precipitate was collectedby filtration washing with water. The precipitate was dissolved in DCM(150 mL) and washed with saturated NaHCO₃ (100 mL), water (100 mL) andbrine (100 mL). The organic portion was dried (MgSO₄) and evaporated togive a white solid. Purification by flash column chromatography[CHCl₃/MeOH 0% to 4% in 1% increments] gave the product as a white solid(0.696 g, 73%). Analytical Data: RT 1.55 min; MS (ES⁺) m/z (relativeintensity) 1096 ([M+H]^(+⋅), 20), 1118 ([M+Na]^(+⋅), 100)

(iv) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-y)oxy)propoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(21)

Pd(PPh₃)₄ (14 mg, 12.28 μmol, 0.02 eq.) was added to a solution of thecyclised product 20 (0.673 g, 0.61 mmol, 1.0 eq.) and pyrrolidine (55mg, 63 μL, 0.8 mmol, 1.25 eq.) in anhydrous DCM (30 mL). The solutionwas stirred at room temperature for 30 min. The reaction mixture wasdiluted with DCM (70 mL) and washed with saturated NH₄Cl (100 mL),saturated brine (100 mL), dried (MgSO₄) and evaporated to give an offwhite foam. The product was triturated with diethyl ether and dried togive the product (0.62 g, 100%) which was used without furtherpurification. Analytical Data: RT 1.16 min; MS (ES⁺) m/z (relativeintensity) 1012 ([M+H]^(+⋅), 80), 1034 ([M+Na]^(+⋅), 20)

(v) tert-butyl(11S,11aS)-8-(3-(((11S,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(22)

EDCl.HCl (0.13 g, 0.66 mmol, 1.1 eq.) was added to a cloudy solution ofcompound 21 (0.61 g, 0.6 mmol, 1.0 eq.) and Mal-dPEG₈@-OH (0.393 g, 0.66mmol, 1.1 eq.) in CHCl₃ (25 mL). The clear solution was stirred at roomtemperature for 1.5h., diluted with CHCl₃ (100 mL) washed with brine(2×100 mL), dried (MgSO₄) and evaporated under reduced pressure to givea yellow foam. Purification by flash column chromatography [CHCl₃/MeOH0% to 6% in 1% increments gave the product as a white foam (0.786 g,82%). Analytical Data: RT 1.44 min; MS (ES⁺) m/z (relative intensity)1586 ([M+H]^(+⋅), 40), 1609 ([M+Na]^(+⋅), 100)

(vi)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(23)

Ice cold 95% TFA_((aq)) solution (10 mL) was added to the Boc protectedcompound 22 (0.759 g, 0.48 mmol, 1.0 eq) which had been cooled to 0° C.(ice bath). The yellow solution was stirred at 0° C. for 1h. Thereaction mixture was poured onto ice/water (200 mL) and the mixture wasbasified to pH 8 with solid NaHCO₃. The mixture was extracted with DCM(4×50 mL) and the combined extracts washed with brine (100 mL), dried(MgSO₄) and evaporated under reduced pressure. The product was purifiedby flash column chromatography [CHCl₃/MeOH 0% to 8% in 1% increments] togive a pale yellow foam (0.445 g, 65%). Analytical Data: RT 1.37 min; MS(ES⁺) m/z (relative intensity) 1468 ([M+H]^(+⋅), 40)

Example 2 Alternate Synthesis of Tert-Butyl(5-(3-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(19)

(i)((2S,2'S)-(4,4′-(propane-1,3-diylbis(oxy))bis(5-methoxy-2-nitrobenzoyl))bis(4-methylenepyrrolidine-1,2-diyl))bis(methylene)diacetate(24)

A solution of acetyl chloride (21.1 mL, 23.3 g, 297 mmol) in DCM (100mL) was added drop-wise over a period of 20 min to a stirred solution ofbis-alcohol (9) (75 g, 114 mmol) and triethylamine (34.7 g, 343 mmol) inanhydrous DCM (900 mL) at 0-5° C. under a nitrogen atmosphere. Thereaction mixture was allowed to warm to room temperature and stirred fora further 60 mins. The reaction mixture was washed with ice cold 0.5MHCl (500 mL), saturated aqueous sodium hydrogen carbonate (250 mL),brine (100 mL) and dried (MgSO₄). Removal of the solvent by rotaryevaporation gave a pale yellow foam which was used in the next stepwithout further purification. Yield=66.7 g (79%). Analytical Data:Purity satisfactory by LC/MS (7.60 min (ES⁺) m/z (relative intensity)741.2 ([M+1]^(+⋅), 60) 763.3 ([M+Na]^(+⋅), 100)); ¹H NMR (400 MHz,CDCl₃) δ 67.73 (s, 2H), 6.83 (s, 2H), 5.12 (d, 2H, J=12 Hz), 5.02 (s,2H), 4.89 (s, 2H), 4.79 (m, 2H), 4.61 (m, 1H), 4.35 (m, 6H), 3.98 (s,6H), 3.87 (d, 1H, J=4.0 Hz), 3.76 (m, 3H), 2.87-2.83 (m, 2H), 2.56-2.43(m, 4H), 2.05 (s, 4H), 1.96 (s, 2H).

(ii)((2S,2'S)-(4,4′-(propane-1,3-diylbis(oxy))bis(2-amino-5-methoxybenzoyl))bis(4-methylenepyrrolidine-1,2-diyl))bis(methylene) diacetate (25)

A 10% solution of formic acid in methanol (500 mL) was added in one go,via a separating funnel, to a solution of the bis-alcohol (24) (66 g,0.09 mol) in methanol (1000 mL) containing zinc* (145 g, 2.22 mol) atroom temperature. The temperature of the reaction mixture rapidly roseto 42° C. and was then cooled back to room temperature with the aid of acold water bath. The excess zinc was removed by filtering through ashort bed of celite, which was then washed with ethyl acetate (100 mL).The filtrate was diluted with ethyl acetate (1400 mL) and washed withsaturated sodium hydrogen carbonate (1500 mL), water (500 mL), brine(100 mL) and dried (MgSO₄). Removal of the solvent by rotary evaporationgave a yellow solid which was purified by column chromatography (4%methanol/DCM) to give the product as a pale yellow foam. Yield=38.1 g(63%). Analytical Data: Purity satisfactory by LC/MS (6.61 min (ES⁺) m/z(relative intensity) 681.2 ([M+1]^(+⋅), 100)); ¹H NMR (400 MHz, CDCl₃)6.74 (s, 2H), 6.31 (s, 2H), 5.02 (bs, 2H), 4.97 (bs, 2H), 4.80 (s, 2H),4.33-4.10 (m, 16H), 3.78 (s, 3H), 2.78 (m, 2H), 2.46 (m, 2H), 2.34 (m,2H), 2.04 (s, 6H).

(iii)((S)-1-(4-(3-(4-((S)-2-(acetoxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-((tert-butoxycarbonyl)amino)-2-methoxyphenoxy)propoxy)-2-amino-5-methoxybenzoyl)-4-methylenepyrrolidin-2-yl)methylacetate (26)

Boc anhydride (21.6 g, 31.7 mmol) was added to a solution of the diamine(25) (6.92 g 31.7 mmol) in THE (200 mL) at room temperature. Theresulting solution was then heated at reflux for 3 hours, cooled andevaporated to dryness under reduced pressure. The resulting residue waspurified by column chromatography (70-100% ethyl acetate/hexane) to givethe product as a pale yellow solid. Yield=8.4 g (34%).Analytical Data:Purity satisfactory by LC/MS (3 min run) (1.64 min (ES⁺) m/z (relativeintensity) 781.2 ([M+Na]^(+⋅), 30)); ¹H NMR (400 MHz, CDCl₃) 8.32 (bs,1H), 7.87 (s, 1H), 6.80 (s, 1H), 6.73 (s, 1H), 5.02 (m, 3H), 4.79 (3H),4.34-4.09 (m, 14H), 3.83 (s, 3H), 3.78 (s, 3H), 2.81-2.74 (m, 2H),2.48-2.36 (m 4H), 2.04 (m 7H), 1.49 (s, 9H).

(iv)((S)-1-(4-(3-(4-((S)-2-(acetoxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-2-methoxyphenoxy)propoxy)-2-((tert-butoxycarbonyl)amino)-5-methoxybenzoyl)-4-methylenepyrrolidin-2-yl)methylacetate (27)

Triethyl amine (0.57 g, 5.6 mmol) was added in one go to a solution ofthe amine (26) (2 g, 2.56 mmol) and triphosgene (0.27 g, 0.92 mmol) inTHE (30 mL) under nitrogen. The resulting mixture was heated at 40° C.for 5 min. A small aliquot was quenched with methanol, and LCMSindicated complete conversion to the methyl carbamate (m/z 983, M+1). Aslurry of SG3366 (2.25 g, 3.48 mmol) and triethyl amine (0.39 g, 3.84mmol) in THE (50 mL) was added in one go and the resulting mixtureheated at 40° C. for 4 hours. After cooling, the white solid was removedby filtration and the filtrate evaporated to dryness under reducedpressure, and purified by column chromatography (1-3% methanol/DCM) togive the product as a pale yellow solid. Yield=2.1 g (69%). AnalyticalData: Purity satisfactory by LC/MS (8.26 min (ES⁺) m/z (relativeintensity) 1184.3 ([M+1]⁺, 70), 1206.3 ([M+Na]^(+⋅), 100)); ¹H NMR (400MHz, DMSO-d₆) δ 7.62 (s, 2H), 7.30 (s, 2H), 4.29 (t, 4H, J=6.0 Hz), 3.85(s, 6H), 2.30-2.26 (m, 2H).

(v)((S)-1-(4-(3-(4-((S)-2-(acetoxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-2-methoxyphenoxy)propoxy)-2-((tert-butoxycarbonyl)amino)-5-methoxybenzoyl)-4-methylenepyrrolidin-2-yl)methylacetate (19)

Potassium carbonate (1.16 g, 8.44 mmol) was dissolved in water (8.4 mL)and added to a solution of the diacetate (27) (2.0 g, 1.69 mmol) inmethanol (40 mL). The resulting mixture was stirred at 25° C. for 30mins, then evaporated to dryness under reduced pressure. The resultingresidue was taken up in water (100 mL), acidified (pH 3) with 1M citricacid and extracted with ethyl acetate (3×100 mL). The combined extractswere washed with water (100 mL), brine (30 mL) and dried (MgSO₄).Removal of the solvent under reduced pressure left the product as anoff-white solid which was used in the next step without furtherpurification. Yield=1.6 g (87%). Analytical Data: Purity satisfactory byLC/MS (7.32 min (ES⁺) m/z (relative intensity) 1100.7 ([M+1]⁺, 50),1122.3 ([M+Na]^(+⋅), 100)); ¹H NMR (400 MHz, DMSO-d₆) δ9.98 (bs, 1H),9.09 (bs, 1H), 8.73 (bs, 1H), 8.14 (d, J=8 Hz, 1H), 7.59 (d, J=8 Hz,2H), 7.33 (d, J=8 Hz, 2H), 7.21 (m, 3H), 6.90 (bs, 2H), 5.91 (m, 1H),5.30 (d, J=4 Hz, 1H), 5.19 (d, J=4 Hz, 1H), 5.00 (m, 6H), 4.70-4.35 (m,6H), 4.15-3.88 (m, 12H), 3.77 (s, 3H), 3.67 (s, 3H), 2.82-2.67 (m, 2H),2.42 (m, 3H), 2.21 (t, J=4 Hz, 2H), 1.98 (m, 6H), 1.42 (s, 9H), 1.31 (d,J=8 Hz, 3H), 0.90 (d, J=4 Hz, 3H), 0.84 (d, J=4 Hz, 3H).

Example 3

(a) Ally(5-(3-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((tert-butyldimethylsllyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(28)

A solution of allyl chloroformate (1.05 g, 0.9 mL, 8.7 mmol, 0.9 eq.)was added drop wise to a solution of bis aniline (11)(8.02 g, 9.7 mmol,1 eq.) and pyridine (1.15 g, 1.2 mL, 14.55 mmol, 1.9 eq.) in dry DCM(350 mL) at −78° C. (dry ice/acetone bath). The reaction mixture wasstirred at −78° C. for 1 hour and then allowed to reach roomtemperature. The reaction mixture was washed with saturated aqueouscopper sulphate solution (250 mL), water (250 mL), saturated sodiumbicarbonate (250 mL), brine (250 mL) and dried (MgSO₄). Rotaryevaporation under reduced pressure afforded the crude product.Purification by flash chromatography 50% n-hexane/50% ethyl acetate, to20% n-hexane/80% ethyl acetate to 100% ethyl acetate to 1% methanol/99%ethyl acetate] gave the bis-alloc product (2.066 g), the desiredmono-alloc product (4.33 g, 49%) and recovered bis-aniline (1.96 g).Analytical Data: RT 2.26 min; MS (ES⁺) m/z (relative intensity) 909([M+1]^(+⋅), 100); 931 ([M+Na]^(+⋅), 100)

(b)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(5-(3-(5-(((allyloxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(29)

Triethylamine (1.22 g, 1.7 mL, 12.1 mmol, 2.2 eq.) was added to astirred solution of the mono-boc protected bis-aniline (28) (5.0 g, 5.5mmol, 1.0 eq.) and triphosgene (0.59 g, 1.98 mmol, 0.36 eq.) in dry THE(75 mL) under a nitrogen atmosphere at room temperature. The reactionmixture was heated to 40° C., after 5 min a sample was treated withmethanol and analysed by LCMS as the methyl carbamate. Analytical Data:RT 2.30 min; MS (ES⁺) m/z (relative intensity) 967 ([M+H]^(+⋅), 25), 989([M+Na]^(+⋅), 100)

A solution/suspension of the benzyl-alcohol (17) (3.11 g, 8.25 mmol, 1.5eq.) and triethylamine (0.83 g, 1.1 mL 2.6 mmol, 1.5 eq.) in dry THE (75mL) was run in from a dropping funnel to the freshly preparedisocyanate. The reaction mixture was stirred at 40° C. for 5h. thenovernight at room temperature The reaction mixture was allowed to cool,filtered and the filtrate evaporated to dryness to afford the crudeproduct as a yellow oil which was purified by flash columnchromatography [50% n-hexane/50% ethyl acetate to 40% n-hexane/60% ethylacetate] which gave the product as a yellow glass (1.25 g, 17%).Analytical Data: RT 2.26 min; MS (ES⁺) m/z (relative intensity) 1312([M+H]^(+⋅), 25), 1335 ([M+Na]^(+⋅), 35)

(c)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(5-(3-(5-(((allyloxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(30)

A 1.0M solution of TBAF in THE (5.2 mL, 5.2 mmol, 2.2 eq.) was added toa solution of the bis TBS compound (29) (3.096 g, 2.36 mmol, 1.0 eq.) inanhydrous THE (25 mL). The mixture was stirred at room temperature for30 min., the solvent was removed under reduced pressure and the residuepurified by flash column chromatography [ethyl acetate/methanol 0% to 6%in 1% increments] which gave the desired product as a white foam (1.91g, 75%). Analytical Data: RT 1.56 min; MS (ES⁺) m/z (relative intensity)1084 ([M+H]^(+⋅), 100), 1106 ([M+Na]^(+⋅), 90)

(d) Allyl(11S,11aS)-8-(3-(((11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(31)

IBX (45 wt %, 2.06 g, 3.3 mmol, 2.4 eq.) was added to a solution of thebis-alcohol 30 (1.49 g, 1.38 mmol, 1.0 eq.) in anhydrous DMSO (40 mL).The solution was stirred at 30° C. for 18h. LCMS analysis indicated thepresence of a small amount of partially cyclised material. A furtherportion of IBX (45 wt %, 0.171 g, 0.275 mmol, 0.2 eq.) was added and thereaction was continued for a further 24h. The reaction mixture waspoured into water (200 mL) and the resultant precipitate was collectedby filtration washing with water. The precipitate was dissolved in DCM(150 mL) and washed with saturated NaHCO₃ (100 mL), water (100 mL) andbrine (100 mL). The organic portion was dried (MgSO₄) and evaporated togive a white solid. Purification by flash column chromatography[CHCl₃/MeOH 0% to 3% in 1% increments] gave the product as a white solid(1.06 g, 72%). Analytical Data: RT 6.88 min; MS (ES⁺) m/z (relativeintensity) 1080 ([M+H]^(+⋅), 50), 1102 ([M+Na]^(+⋅), 100)

(e) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(32)

Pd(PPh₃)₄ (44 mg, 38.5 μmol, 0.04 eq.) was added to a solution of thecyclised product 31 (1.04 g, 0.96 mmol, 1.0 eq.) and pyrrolidine (0.171mg, 196 pL, 2.4 mmol, 2.5 eq.) in anhydrous DCM (30 mL). The solutionwas stirred at room temperature for 30 min. The reaction mixture wasdiluted with DCM (30 mL) and washed with saturated NH₄Cl (100 mL),saturated brine (100 mL), dried (MgSO₄) and evaporated to give an offwhite foam. The product was triturated with diethyl ether and dried togive the product (0.86 g, 100%) which was used without furtherpurification. Analytical Data: RT 1.10 min; MS (ES⁺) m/z (relativeintensity) 894 ([M+H]^(+⋅), 30)

(f)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(23)

EDCl.HCl (0.203 g, 1.06 mmol, 1.1 eq.) was added to a solution ofcompound 32 (0.86 g, 0.96 mmol, 1.0 eq.) and Mal-dPEG₈@-OH (0.57 g, 0.96mmol, 1.1 eq.) in dry DCM (30 mL) and CHCl₃ (to give a clear solution).The clear solution was stirred at room temperature for 18h. then afurther portion of EDCl.HCl (0.037 g, 0.19 mmol, 0.2 eq.) was added andreaction continued for a further 24h. The reaction mixture was dilutedwith DCM (70 mL) washed with water (100 mL), brine (100 mL), dried(MgSO₄) and evaporated under reduced pressure to give a yellow foam.Purification by flash column chromatography [CHC₃/MeOH 0% to 6% in 1%increments] gave the product as an off white foam (0.56 g, 40%).Analytical Data: RT 6.13 min; MS (ES⁺) m/z (relative intensity) 1468([M+H]^(+⋅), 20)

Example 4

(a) Allyl(2-((R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-(3-(4-((R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-((((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)amino)-2-methoxyphenoxy)propoxy)-4-methoxyphenyl)carbamate(34)

Triethylamine (0.049 g, 0.07 mL, 0.48 mmol, 2.2 eq.) was added to astirred solution of the mono-alloc protected bis-aniline (23) (0.2 g,0.22 mmol, 1.0 eq.) and triphosgene (0.024 g, 0.079 mmol, 0.36 eq.) indry THE (5 mL) under an argon atmosphere at room temperature. Thereaction mixture was heated to 40° C., after 5 min a sample was treatedwith methanol and analysed by LCMS as the methyl carbamate. AnalyticalData: RT 2.27 min; MS (ES⁺) m/z (relative intensity) 967 ([M+H]^(+⋅),80), 989 ([M+Na]^(+⋅), 100)

A solution/suspension of the benzyl-alcohol (33) (0.121 g, 0.29 mmol,1.3 eq.) and triethylamine (0.029 g, 0.04 mL 0.29 mmol, 1.3 eq.) in dryTHE (5 mL) was run in from a dropping funnel to the freshly preparedisocyanate. The reaction mixture was stirred at 40° C. for 4h. thenovernight at room temperature The reaction mixture was allowed to cool,filtered and the filtrate evaporated to dryness to afford the crudeproduct which was purified by flash column chromatography [BiotageIsolera™ CHCl₃/MeOH 2% to 4%, gradient elution]. This gave the product(0.237 g, 79%). Analytical Data: RT 2.19 min; MS (ES⁺) m/z (relativeintensity) 1358 ([M+H]^(+⋅), 30), 1380 ([M+Na]^(+⋅), 15)

(b)4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl(5-(3-(5-amino-4-((R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)propoxy)-2-((R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate(35)

Pd(PPh₃)₄ (0.3 g, 0.25 mmol, 0.06 eq.) was added to a solution of thealloc protected intermediate 34 (5.89 g, 4.3 mmol, 1.0 eq.) andpyrrolidine (0.46 g, 530 μL, 6.5 mmol, 1.5 eq.) in anhydrous DCM (50mL). The solution was stirred at room temperature for 1h. The reactionmixture was diluted with DCM and washed with saturated NH₄Cl, saturatedbrine, dried (MgSO₄) and evaporated to give crude product. The productwas purified by flash column chromatography [Biotage Isolera™ DCM/MeOH1% to 3%] to give the product (4.53 g, 83%) which had an overall purityof 80% and was used without further purification. Analytical Data: RT2.10 min; MS (ES⁺) m/z (relative intensity) 1275 ([M+H]^(+⋅), 40).

(c)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-(3-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-((((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)amino)-2-methoxyphenoxy)propoxy)-4-methoxyphenyl)carbamate(36)

Triethylamine (0.35 g, 48 μL, 0.34 mmol, 2.2 eq.) was added to a stirredsolution of the aniline (35) (0.2 g, 0.157 mmol, 1.0 eq.) andtriphosgene (0.017 g, 57 μmol, 0.36 eq.) in dry THE (5 mL) under anargon atmosphere at room temperature. The reaction mixture was heated to40° C., after 5 min a sample was treated with methanol and analysed byLCMS as the methyl carbamate. Analytical Data: RT 2.15 min; MS (ES⁺) m/z(relative intensity) 1333 ([M+H]^(+⋅), 40), 1354 ([M+Na]^(+⋅), 35).

A solution/suspension of the benzyl-alcohol (17) (0.071 g, 0.19 mmol,1.2 eq.) and triethylamine (19 mg, 26 μL 0.19 mmol, 1.2 eq.) in dry THE(5 mL) was run in from a dropping funnel to the freshly preparedisocyanate. The reaction mixture was stirred at 40° C. for 4h. thenovernight at room temperature The reaction mixture was filtered and thefiltrate evaporated to dryness to afford the crude product which waspurified by flash column chromatography [Biotage Isolera™ CHCl₃/MeOH 2%to 3%, gradient elution] which gave the product (0.152 g, 58%).Analytical Data: RT 2.12 min; MS (ES⁺) m/z (relative intensity) 1677([M+H]^(+⋅), 30), 1700 ([M+Na]^(+⋅), 100).

(d)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-(3-(4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-((((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)amino)-2-methoxyphenoxy)propoxy)-4-methoxyphenyl)carbamate(37)

A 1.0M solution of TBAF in THE (3.4 mL, 3.4 mmol, 2.0 eq.) was added toa solution of the bis TBS compound (36) (2.86 g, 1.7 mmol, 1.0 eq.) inanhydrous THE (30 mL) under an argon atmosphere. The mixture was stirredat room temperature for 6h., the reaction mixture was diluted with CHCl₃and washed with water, brine, dried (MgSO₄) and evaporated under reducedpressure to give a yellow solid. The residue was purified by flashcolumn chromatography [Biotage Isolera™ CHCl₃/MeOH, gradient elutionwith the product eluting at 4% MeOH] which gave the desired product(1.365 g) and mixed fractions which were further purified by flashcolumn chromatography [CHCl₃/MeOH 1% to 5%] to give further product(0.562 g) this gave a combined yield of desired product (1.93 g, 75%).Analytical Data: RT 1.55 min; MS (ES⁺) m/z (relative intensity) 1449([M+1]^(+⋅), 25); 1471 ([M+Na]^(+⋅), 20).

(e)4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-11-hydroxy-8-(3-(((S)-10-(((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(38)

IBX (45 wt %, 0.236 g, 0.38 mmol, 2.2 eq.) was added to a solution ofthe bis-alcohol 37 (0.25 g, 0.17 mmol, 1.0 eq.) in anhydrous DMSO (12mL). The solution was stirred at 30° C. for 3.5d. The reaction mixturewas poured into water (100 mL) and the resultant precipitate wascollected by filtration washing with water. The precipitate wasextracted with DCM (5×30 mL) and the combined fractions were washed withsaturated NaHCO₃ (60 mL), water (60 mL) and brine (60 mL). The organicportion was dried (MgSO₄) and evaporated to give crude product.Purification by flash column chromatography [CHCl₃/MeOH 1% to 5%] gavethe product as a white solid (0.158 g, 64%). Analytical Data: RT 1.53min; MS (ES⁺) m/z (relative intensity) 1445 ([M+1]*-, 20); 1467([M+Na]^(+⋅), 30).

(f) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-11-hydroxy-8-(3-(((11S,11aS)-11-hydroxy-10-(((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-y)oxy)propoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(39)

Pd(PPh₃)₄ (8 mg, 6.9 μmol, 0.06 eq.) was added to a solution of thecyclised product 38 (0.158 g, 0.109 mmol, 1.0 eq.) and pyrrolidine (0.01g, 12 μL, 0.15 mmol, 1.5 eq.) in anhydrous DCM (10 mL). The solution wasstirred at room temperature for 15 min. The reaction mixture was dilutedwith CHCl₃ and washed with saturated sodium bicarbonate solution,saturated brine, dried (MgSO₄) and evaporated to give crude product. Theproduct was triturated with diethyl ether (×3) and dried to give theproduct (0.136 g, 100%) which was used without further purification.Analytical Data: RT 1.21 min; MS (ES⁺) m/z (relative intensity) 1361([M+1]^(+⋅), 50); 1384 ([M+Na]^(+⋅), 10).

(g)4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-8-(3-(((11S,11aS)-11-hydroxy-10-(((4-((10S,13S)-O-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-y)oxy)propoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate(40)

A solution of compound 39 (0.136 g, 0.1 mmol, 1.0 eq.), Mal-dPEG₈@-OH(0.066 g, 0.11 mmol, 1.1 eq.) and EDCI.HCl (0.022 g, 0.11 mmol, 1.1 eq.)in dry DCM (10 mL) and MeOH (1 drop) was stirred at room temperature for1.45h. The reaction mixture was diluted with CHCl₃ and washed withwater, brine, dried (MgSO₄) and evaporated under reduced pressure togive the crude product. Purification by flash column chromatography[CHCl₃/MeOH 1% to 9%] gave the product as a white solid (0.123 g, 63%).Analytical Data: [a]_(D)=+112.5° (c=0.4, hplc CHCl₃); RT 6.37 min; MS(ES⁺) m/z (relative intensity) 1936 ([M+1]^(+⋅), 35); 1958 ([M+Na]^(+⋅),15).

Example 5—Conjugation

Conjugate Trastuzumab-23

A 50 mM solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP)in phosphate-buffered saline pH 7.4 (PBS) was added (50 molarequivalent/antibody, 35 micromoles, 700 μL) to a 24.14 mL solution ofantibody, trastuzumab, (105 mg, 700 nanomoles) in reduction buffercontaining PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and afinal antibody concentration of 4.35 mg/mL. The reduction mixture washeated at +37° C. for 3 hours (or until full reduction is observed byUHPLC) in an incubator with gentle (<150 rpm) shaking. After coolingdown to room temperature, the reduced antibody was buffer exchanged, viaspin filter centrifugation, into a reoxidation buffer containing PBS pH7.4 and 1 mM EDTA to remove all the excess reducing agent. A 50 mMsolution of dehydroascorbic acid (DHAA, 10 molar equivalent/antibody, 7micromoles, 140 μL) in DMSO was added and the reoxidation mixture wasallowed to react for 16 hours at room temperature with gentle (<150 rpm)shaking at an antibody concentration of 2.3 mg/mL (or more DHAA addedand reaction left for longer until full reoxidation of the cysteinethiols to reform the inter-chain cysteine disulfides is observed byUHPLC). The reoxidation mixture was then sterile-filtered and diluted ina conjugation buffer containing PBS pH 7.4, 1 mM EDTA for a finalantibody concentration of 1.0-1.5 mg/mL. Compound 23 (SG3400) was addedas a DMSO solution (10 molar equivalent/antibody, 1 micromole, in 1.0 mLDMSO) to 9 mL of this reoxidised antibody solution (15 mg, 100nanomoles) for a 10% (v/v) final DMSO concentration. The solution wasmixed for 1.5 hours at room temperature, then the conjugation wasquenched by addition of N-acetyl cysteine (4 micromoles, 40 L at 100mM), diluted to >50 mL in PBS and conjugate trastuzumab-23 was purifiedby spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spinfilter, sterile-filtered and analysed. UHPLC analysis on a ShimadzuProminence system using a Phenomenex Aeris 3.6u XB-C18 150 mm×2.1 mmcolumn eluting with a gradient of water and acetonitrile on a reducedsample of conjugate trastuzumab-A at 280 nm and 330 nm (Compound Aspecific) shows unconjugated light chains and a mixture of unconjugatedheavy chains and heavy chains attached to a single molecule of compound23, consistent with a drug-per-antibody ratio (DAR) of 1.71 molecules ofcompound 23 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel G3000SWXL 5 μm 7.8×300 mm column (with a 7 μm 6.0×40 mm guardcolumn) eluting with sterile-filtered SEC buffer containing 200 mMpotassium phosphate pH 6.95, 250 mM potassium chloride and 10%isopropanol (v/v) on a sample of conjugate trastuzumab-23 at 280 nmshows a monomer purity of 94%. UHPLC SEC analysis gives a concentrationof final conjugate trastuzumab-23 at 0.84 mg/mL in 15 mL, obtained massof conjugate trastuzumab-23 is 12.7 mg (84% yield).

Conjugate Trastuzumab-40

A 50 mM solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP)in phosphate-buffered saline pH 7.4 (PBS) was added trastuzumab, (50molar equivalent/antibody, 50 micromoles, 1.0 mL) to a 34.5 mL solutionof antibody (150 mg, 1.0 micromole) in reduction buffer containing PBSand 1 mM ethylenediaminetetraacetic acid (EDTA) at a final antibodyconcentration of 4.35 mg/mL. The reduction mixture was heated at +37° C.for 3 hours (or until full reduction is observed by UHPLC) in anincubator with gentle (<150 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filtercentrifugation, into a reoxidation buffer containing PBS and 1 mM EDTAto remove excess reducing agent. A 50 mM solution of dehydroascorbicacid (DHAA, 50 molar equivalent/antibody, 50 micromoles, 1.0 mL) in DMSOwas added and the reoxidation mixture was allowed to react for 2 hoursat room temperature (or until full reoxidation of the cysteine thiols toreform the inter-chain cysteine disulfides is observed by UHPLC) withgentle (<150 rpm) shaking at an antibody concentration of 2-3 mg/mL. Thereoxidation mixture was then sterile-filtered and diluted in aconjugation buffer containing PBS and 1 mM EDTA to a final antibodyconcentration of ˜1.5 mg/mL. Compound 40 was added as a DMSO solution(10 molar equivalent/antibody, 1 micromole, in 0.9 mL DMSO) to 9 mL ofthis reoxidised antibody solution (15 mg, 100 nanomoles). The solutionwas mixed for 1.25 hours at room temperature, after which theconjugation reaction was quenched by addition of N-acetyl cysteine (4micromoles, 40 L at 100 mM) and diluted to >50 mL in PBS. Theconjugation mixture was purified by spin filtration using a 15 mL AmiconUltracell 50 kDa MWCO spin filter, sterile-filtered, analysed and storedat +4° C. The reduction and reoxidation steps are monitored bycomparison of the relative amounts of individual light and heavy chainswith full length antibody as observed by UHPLC analysis on a ShimadzuProminence system using a Phenomenex Aeris 3.6u XB-C18 150×2.1 mm columneluting with a gradient of water and acetonitrile. UHPLC analysis on aShimadzu Prominence system using a Phenomenex Aeris 3.6u XB-C18 150×2.1mm column eluting with a gradient of water and acetonitrile on a reducedsample of conjugate trastuzumab-B at 280 nm and 330 nm (Compound 40specific) shows unconjugated light chains and a mixture of unconjugatedheavy chains and heavy chains attached to a single molecule of Compound40, consistent with a drug-per-antibody ratio (DAR) of 1.68 molecules ofCompound 40 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of conjugate trastuzumab-40 at 280nm shows a monomer purity of 93% with no impurity detected. UHPLC SECanalysis gives a concentration of final conjugate trastuzumab-B at 0.74mg/mL in 17 mL, obtained mass of conjugate trastuzumab-40 is 12.5 mg(83% yield).

Conjugate R347-23

A 50 mM solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP)in phosphate-buffered saline pH 7.4 (PBS) was added (42 molarequivalent/antibody, 56 micromoles, 1.12 mL at 50 mM) to a 14.09 mLsolution of antibody (200 mg, 1.33 micromoles) in reduction buffercontaining PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and afinal antibody concentration of 4.0 mg/mL. The reduction mixture washeated at +25° C. for 24 hours (or until full reduction observed byUHPLC) in an incubator with gentle (<100 rpm) shaking. After coolingdown to room temperature, the reduced antibody was buffer exchanged, viaTangential Flow Filtration unit (TFF) using mPES, MidiKros® 30 kDa fiberfilter with 115 cm² surface area, into a reoxidation buffer containingPBS pH 7.4 and 1 mM EDTA to remove all the excess reducing agent. Thereduced antibody was centrifuged for 3 min at 4000 rpm and then filteredusing 0.45 pM membrane filter. A 50 mM solution of dehydroascorbic acid(DHAA, 15 molar equivalent/antibody, 20 micromoles, 400 μL at 50 mM) inDMSO was added and the reoxidation mixture was allowed to react for 16hours at room temperature with gentle (<100 rpm) shaking at an antibodyconcentration of 2.5 mg/mL (or until full reoxidation of the cysteinethiols to reform the inter-chain cysteine disulfides is observed byUHPLC). The reoxidation mixture was centrifuged for 3 min at 4000 rpmand then sterile-filtered using 0.2 pM membrane filter. Compound 23 wasadded as a DMSO solution (10 molar equivalent/antibody, 13.3 micromoles,in 6.6 mL DMSO) to 80 mL of this reoxidised antibody solution (200 mg,1.33 micromoles) for a 10% (v/v) final DMSO concentration. The solutionwas shaken for 3 hours at +25° C. and then the conjugation was quenchedwith N-acetyl cysteine (72.3 micromoles, 0.72 mL at 100 mM).

Excess free drug was removed via Tangential Flow Filtration unit (TFF)using mPES, MidiKros® 30 kDa fiber filter with 115 cm² surface area,into buffer containing PBS pH 7.4. Extent of free drug removal wasmonitored by UHPLC-RP using neat conjugate. After complete removal offree drug, ADC were formulated onto 25 mM Histidine, 200 mM Sucrose, pH6.0, via TFF using mPES, MidiKros® 30 kDa fiber filter with 115 cm²surface area. The whole process of R347 conjugation with Compound 23 wasrepeated with 400 mg antibody and also purified using TFF. ADC from bothbatches were combined and then filtered using Mustang filter understerile atmosphere and then further stored at −78° C.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm(Compound 23 specific) shows a mixture of light and heavy chainsattached to several molecules of Compound 23, consistent with adrug-per-antibody ratio (DAR) of 1.71 molecules of Compound 23 perantibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomerpurity of greater than 97%. UHPLC SEC analysis gives a concentration offinal ADC at 1.92 mg/mL in 265 mL, obtained mass of ADC is 509 mg (85%yield).

Conjugate R347-40

A 50 mM solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP)in phosphate-buffered saline pH 7.4 (PBS) was added (50 molarequivalent/antibody, 20 micromoles, 0.4 mL) to a 13.25 mL solution ofantibody (60 mg, 0.4 micromoles) in reduction buffer containing PBS and1 mM ethylenediaminetetraacetic acid (EDTA) at a final antibodyconcentration of 4.5 mg/mL. The reduction mixture was heated at +37° C.for 3 hours (or until full reduction is observed by UHPLC) in anincubator with gentle (<150 rpm) shaking. After cooling down to roomtemperature, the reduced antibody was buffer exchanged, via spin filtercentrifugation, into a reoxidation buffer containing PBS and 1 mM EDTAto remove excess reducing agent. A 50 mM solution of dehydroascorbicacid (DHAA, 12 molar equivalent/antibody, 4.8 micromoles, 96 μL) in DMSOwas added and the reoxidation mixture was allowed to react for 17 hoursat room temperature (or until full reoxidation of the cysteine thiols toreform the inter-chain cysteine disulfides is observed by UHPLC) withgentle (<150 rpm) shaking at an antibody concentration of ˜1.6 mg/mL.The reoxidation mixture was then sterile-filtered and diluted in aconjugation buffer containing PBS and 1 mM EDTA to a final antibodyconcentration of ˜1.5 mg/mL. Compound 40 was added as a DMSO solution(11 molar equivalent/antibody, 0.44 micromoles, in 0.45 mL DMSO) to 4.05mL of this reoxidised antibody solution (6 mg, 40 nanomoles). Thesolution was mixed for 1.25 hours at room temperature, after which theconjugation reaction was quenched by addition of N-acetyl cysteine (1.76micromoles, 17.6 μL at 100 mM). The conjugation mixture was purified byspin filtration with PBS using a 15 mL Amicon Ultracell 50 kDa MWCO spinfilter, sterile-filtered, analysed and stored at +4° C.

The reduction and reoxidation steps are monitored by comparison of therelative amounts of individual light and heavy chains with full lengthantibody as observed by UHPLC analysis on a Shimadzu Prominence systemusing a Phenomenex Aeris 3.6u XB-C18 150×2.1 mm column eluting with agradient of water and acetonitrile. UHPLC analysis on a ShimadzuProminence system using a Phenomenex Aeris 3.6u XB-C18 150×2.1 mm columneluting with a gradient of water and acetonitrile on a reduced sample ofConjugate R347-40 at 280 nm and 330 nm (Conjugate 40 specific) showsunconjugated light chains and a mixture of unconjugated heavy chains andheavy chains attached to a single molecule of Compound 40, consistentwith a drug-per-antibody ratio (DAR) of 1.86 molecules of Compound 40per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel G3000SWXL 5 μm 7.8×300 mm column (with a 7 μm 6.0×40 mm guardcolumn) eluting with sterile-filtered SEC buffer containing 200 mMpotassium phosphate pH 6.95, 250 mM potassium chloride and 10%isopropanol (v/v) on a sample of Conjugate R347-40 at 280 nm shows amonomer purity of 97% with no impurity detected. UHPLC SEC analysisgives a concentration of final Conjugate R347-40 at 0.87 mg/mL in 5.5mL, obtained mass of Conjugate R347-40 is 4.8 mg (80% yield).

Conjugate HLL2-23

A 50 mM solution of DTT (Dithiothreitol) in phosphate-buffered saline pH7.4 (PBS) was added (40 molar equivalent/antibody, 40 micromoles, 825μL) to a 37.5 mL solution of antibody HLL2 (150 mg, 1 micromol) inreduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid(EDTA) and a final antibody concentration of 4 mg/mL. The reductionmixture was incubated at room temperature overnight with gentle (135rpm) shaking. The reduced antibody was buffer-exchanged against PBS+1 mMEDTA (to remove the excess of DTT) using TFF (Tangential FlowFiltration, Spectrum Labs 115 cm² hollow fibre cassette with 50 kDamolecular weight cut off). The sample was filtered using a 0.4 μmsyringe filter to remove any debris from the TFF step and antibodyconcentration brought to 1.5 mg/mL before reoxidation. A 50 mM solutionof dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 13.9micromoles, 0.28 mL) in DMSO was added and the reoxidation mixture wasallowed to react for 16 hours at room temperature under gentle (<150rpm) shaking. The reoxidation mixture was then sterile-filtered; 139 mgof antibody (92.6 mL as 1.5 mg/mL solution) was obtained, 14 mL of whichwas taken forward for conjugation with Compound 23 (estimated ca. 21 mgantibody, 0.14 micromoles). Compound 23 was added as a DMSO solution (10molar equivalent/antibody, 0.33 micromoles in 0.133 mL DMSO) to 14 mL ofthe reoxidised antibody solution. The conjugation mixture was toppedwith 1.27 ml of DMSO to bring the final DMSO concentration to 10% (v/v)and incubated for 3 hours at room temperature under gentle agitation(135 rpm). Free drug was then removed from the antibody-drug conjugateby extensive diafiltration in PBS using a spin filter device (AmiconUltra-30K centrifugal filter, Millipore). The resulting conjugationmixture was sterile-filtered and analysed by UHPLC.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 214 nm (ADC) and 330 nm(Compound 23 specific) shows a mixture of heavy chains eitherunconjugated or attached to 1 molecule of Compound 23, consistent with adrug-per-antibody ratio (DAR) of 1.64 molecules of Compound 23 perantibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomerpurity greater than 97%. UHPLC SEC analysis gives a concentration offinal ADC at 2.06 mg/mL in 10 mL, obtained mass of ADC is 20.6 mg.

Conjugate AntiCD79b-23

A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered salinepH 7.4 (PBS) was added (80 molar equivalent/antibody, 53.3 micromoles,1.07 mL) to a 25 mL solution of antibody CD79b (100 mg, 667 nmol) inreduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid(EDTA) and a final antibody concentration of 4 mg/mL. The reductionmixture was allowed to react at room temperature overnight with gentleshaking. The reduced antibody was buffer exchanged, via spin filtercentrifugation, into a reoxidation buffer containing PBS and 1 mM EDTAto remove all the excess reducing agent. A 50 mM solution ofdehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 9.28micromoles, 185 μL) in DMSO was added and the reoxidation mixture wasallowed to react for 16 hours at room temperature under gentle (<150rpm) shaking. The reoxidation mixture was then sterile-filtered;Compound 23 was added as a DMSO solution (15 molar equivalent/antibody,1.8 micromoles in 1.0 mL DMSO) to 9 mL of this reoxidised antibodysolution (18 mg, 120 nanomoles) for a 10% (v/v) final DMSO concentrationand a final antibody concentration of 1.8 mg/mL in PBS+1 mM EDTA. Thesolution was mixed for 2 hours at room temperature under gentleagitation (135 rpm), then the conjugation was quenched by addition ofN-acetyl cysteine (7.2 micromoles, 72 L at 100 mM), then purified byspin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter,sterile-filtered and analysed.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris3.6u XB-C18 150×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of Conjugate at 280 nm (ADC) and 330 nm(Compound 23 specific) unconjugated light chains and a mixture ofunconjugated heavy chains and heavy chains attached to 1 or 2 moleculesof Compound 23, consistent with a drug-per-antibody ratio (DAR) of 2.08molecules of Compound 23 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ADC at 280 nm shows a monomerpurity of greater than 98%. UHPLC SEC analysis gives a concentration offinal ADC at 1.62 mg/mL in 7.6 mL, obtained mass of ADC is 12.28 mg.

Example 5—In Vitro Testing

Medium from sub-confluent (80-90% confluency) cell culture in a T75flask was aspirated and the flask rinsed with PBS (about 20 ml) andemptied. Trypsin-EDTA (5 ml) was added, the flask returned to the 37° C.gassed incubator for up to about 5 minutes, then rapped sharply todislodge and dissociate cells from the plastic. The cell suspension wastransferred to a sterile 50 ml screw-top centrifuge tube, diluted withgrowth medium to a final volume of 15 ml, then centrifuged (400 g for 5min). The supernatant was aspirated and the pellet re-suspended in 10 mlculture medium. Repeated pipetting may be necessary to producemonodisperse cell suspensions. The cell concentration and viability aremeasured of trypan blue cell stained cells, using a haemocytometer.Cells were diluted to 2×10⁵/ml, dispensed (50 μl/well) into 96 well flatbottom plates and incubated overnight before use.

A stock solution (1 ml) of antibody drug conjugate (ADC) (20 pg/ml) wasmade by dilution of filter-sterilised ADC into cell culture medium. Aset of 8×10-fold dilutions of stock ADC were made in a 24 well plate byserial transfer of 100 μl onto 900 μl of cell culture medium.

ADC dilution was dispensed (50 μl/well) into 4 replicate wells of the96-well plate, containing 50 μl cell suspension seeded the previous day.Control wells received 50 μl cell culture medium.

The 96-well plate containing cells and ADCs was incubated at 37° C. in aCO₂-gassed incubator for the exposure time.

At the end of the incubation period, cell viability was measured by MTSassay. MTS (Promega) was dispensed (20 μl per well) into each well andincubated for 4 hours at 37° C. in the CO₂-gassed incubator. Wellabsorbance was measured at 490 nm. Percentage cell survival wascalculated from the mean absorbance in the 4 ADC-treated wells comparedto the mean absorbance in the 4 control untreated wells (100%). IC₅₀ wasdetermined from the doses-response data using GraphPad Prism using thenon-linear curve fit algorithm: sigmoidal, 4PL X is log(concentration).

Cell ADC Cell growth Line Description Exposure medium SKBR3 Breast 4days McCoys with carcinoma Glutamax, 10% FBS BT474 Breast 5 days DMEMwith carcinoma glutamax, 10% FBS NCIN87 Gastric 7 days RPMI 1640 withcarcinoma glutamax, 10% FBS IC₅₀ (μg/ml) in: ADC DAR BT474 NCI-N87 SKBR3trastuzumab-40 1.68 >1 0.019 0.019 trastuzumab-23 1.71 >1 0.030 0.022

Testing of AntiCD79b-23

The concentration and viability of cells from a sub-confluent (80-90%confluency) T75 flask are measured by trypan blue staining, and countedusing the LUNA-II™ Automated Cell Counter. Cells were diluted to2×10⁵/ml, dispensed (50 μl/well) into 96-well flat-bottom plates.

A stock solution (1 ml) of antibody drug conjugate (ADC) (20 pg/ml) wasmade by dilution of filter-sterilised ADC into cell culture medium. Aset of 8×10-fold dilutions of stock ADC were made in a 24-well plate byserial transfer of 100 μl into 900 μl of cell culture medium. ADCdilution was dispensed (50 μl/well) into 4 replicate wells of the96-well plate, containing 50 μl cell suspension seeded the previously.Control wells received 50 μl cell culture medium. The 96-well platecontaining cells and ADCs was incubated at 37° C. in a CO₂-gassedincubator for the exposure time.

At the end of the incubation period, cell viability was measured by MTSassay. MTS (Promega) was dispensed (20 μl per well) into each well andincubated for 4 hours at 37° C. in the CO₂-gassed incubator. Wellabsorbance was measured at 490 nm. Percentage cell survival wascalculated from the mean absorbance in the 4 ADC-treated wells comparedto the mean absorbance in the 4 control untreated wells (100%). IC₅₀ wasdetermined from the dose-response data using GraphPad Prism using thenon-linear curve fit algorithm: sigmoidal dose-response curve withvariable slope.

ADC incubation times were 4 days with WSUDLCL2 (B-cell non-Hodgkinlymphoma) and SUDHL4 (B-lymphocyte), 5 days for Granta519 (B-cellnon-Hodgkin lymphoma) and 6 days for BJAB (Burkitt lymphoma). WSUDLCL2and SUDHL4 were cultured in RPMI 1640 with Glutamax+10% (v/v) HyClone™Fetal Bovine Serum, Granta519 in DMEM+Glutamax with 10% (v/v) HyClone™Fetal Bovine Serum and BJAB in RPMI 1640+Glutamax with 20% (v/v)HyClone™ Fetal Bovine Serum.

EC₅₀ (μg/ml) in: ADC SUDHL4 WSUDLCL2 GRANTA519 BJAB AntiCD79b-23 0.053870.9268 0.04957 0.003158

Example 6

Mice

Female severe combined immune-deficient mice (Fox Chase SCID®,C.B-17/lcr-Prkdcscid, Charles River) were ten weeks old with a bodyweight (BW) range of 16.2 to 21.9 grams on Day 1 of the study. Theanimals were fed adlibitum water (reverse osmosis, 1 ppm Cl), and NIH 31Modified and Irradiated Lab Diet@ consisting of 18.0% crude protein,5.0% crude fat, and 5.0% crude fibre. The mice were housed on irradiatedEnricho'Cobs™ Laboratory Animal Bedding in static micro-isolators on a12-hour light cycle at 20-22° C. (68-72° F.) and 40-60% humidity. CRDiscovery Services specifically complies with the recommendations of theGuide for Care and Use of Laboratory Animals with respect to restraint,husbandry, surgical procedures, feed and fluid regulation, andveterinary care. The animal care and use program at CR DiscoveryServices is accredited by the Association for Assessment andAccreditation of Laboratory Animal Care International (AAALAC), whichassures compliance with accepted standards for the care and use oflaboratory animals.

In Vivo Implantation and Tumour Growth

Xenografts were initiated with BT474 human breast carcinomas maintainedat CR Discovery Services by serial subcutaneous transplantation into theSCID mice (see above). On the day of tumour implant, each test mousereceived a 1-mm³ BT474 fragment implanted subcutaneously in the rightflank, and tumour growth was monitored as the average size approachedthe target range of 100 to 150 mm³. Thirty-three days after tumourimplantation, designated as Day 1 of the study, the animals were sortedinto nine groups each consisting of ten mice with individual tumourvolumes of 75 to 144 mm³ and group mean tumour volumes of 111 to 112mm³. Tumours were measured in two dimensions using calipers, and volumewas calculated using the formula:

Tumour Volume (mm³)=0.5(w²×l)

where w=width and l=length, in mm, of the tumour. Tumour weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumourvolume.

Treatment 1

Treatment began on Day 1 in groups of mice (n=10) with establishedsubcutaneous BT474 tumors (75-196 mm³). Trastuzumab-23 was administeredintravenously once on Day 1 (qd×1) at two dosages (0.3 and 1 mg/kg). Avehicle-treated group served as the control group for efficacy analysis.Tumors were measured twice per week until the study was ended on Day 60.Each mouse was euthanized when its tumor reached the endpoint volume of800 mm³ or on the final day, whichever came first. The time to endpoint(TTE) was calculated for each mouse.

Treatment outcome was determined from percent tumor growth delay (%TGD), defined as the percent increase in median TTE for treated versuscontrol mice, with differences between groups deemed statisticallysignificant at P 5 0.05 using log rank survival analysis. Mice weremonitored for complete regression (CR) and partial regression (PR)responses.

Treatment tolerability was assessed by body weight measurements andfrequent observation for signs of treatment-related side effects.Treatment tolerability was assessed by body weight measurements andfrequent observation for signs of treatment-related side effects. Allregimens were acceptably tolerated.

The median TTE for vehicle-treated controls was 44.4 days, establishinga maximum possible TGD of 15.6 days (35%) for the 60-day study. ADCregimens resulted in the maximum possible TGD, produced survival benefitthat was statistically significantly different from vehicle-treatedcontrols (P<0.01) and could not be distinguished based on log rankanalysis (P>0.05). Differences within ADC treatments were only evidentin the MTV on the final day and numbers and types of regressionresponses produced by each regimen.

Trastuzumab-23 at 1 mg/kg produced four partial regressions (PRs). Theresults are illustrated in FIG. 1.

Treatment 2

Treatment began on Day 1 in groups of mice (n=9 or 10) with establishedsubcutaneous BT474 tumors (108-196 mm³). Trastuzumab-40 was administeredintravenously once on Day 1 (qd×1) at two dosages (0.3 and 1 mg/kg). Avehicle-treated group served as the control group for efficacy analysis.Tumors were measured twice per week until the study was ended on Day 62.Each mouse was euthanized when its tumor reached the endpoint volume of1000 mm³ or on the final day, whichever came first. The time to endpoint(TTE) was calculated for each mouse.

Treatment outcome was determined from percent tumor growth delay (%TGD), defined as the percent increase in median TTE for treated versuscontrol mice, with differences between groups deemed statisticallysignificant at P 5 0.05 using log rank survival analysis. Mice weremonitored for complete regression (CR) and partial regression (PR)responses.

Treatment tolerability was assessed by body weight measurements andfrequent observation for signs of treatment-related side effects.Treatment tolerability was assessed by body weight measurements andfrequent observation for signs of treatment-related side effects.

All regimens were acceptably tolerated. The median TTE forvehicle-treated controls was 52.9 days, establishing a maximum possibleTGD of 9.1 days (17%) for the 62-day study. ADC treatment resulted inthe maximum possible TGD, however only the 1 mg/kg treatment producedsurvival benefit that was statistically significantly different fromvehicle-treated controls (P<0.001).

Trastuzumab-40, at 1 mg/kg produced four partial regressions (PRs) andone complete regression (CR) which remained a tumour-free survivor (TFS)at study end. The results are shown in FIG. 2.

Example 7

Tumor Cell Culture

Human NCI-N87 gastric carcinoma lymphoma cells were cultured inRPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mMglutamine, 100 units/mL penicillin, 100 pg/mL streptomycin sulfate and25 pg/mL gentamicin. The cells were grown in tissue culture flasks in ahumidified incubator at 37° C., in an atmosphere of 5% C02 and 95% air.

In Vivo Implantation and Tumor Growth

The NCI-N87 cells used for implantation were harvested during log phasegrowth and resuspended in phosphate buffered saline (PBS) containing 50%Matrigel™ (BD Biosciences). On the day of tumor implant, each test mouse(SCID mice as in Example 6) was injected subcutaneously in the rightflank with 1×10⁷ cells (0.1 mL cell suspension), and tumor growth wasmonitored as the average size approached the target range of 100 to 150mm³. Eleven days later, designated as Day 1 of the study, mice weresorted according to calculated tumor size into eleven groups eachconsisting of ten animals with individual tumor volumes ranging from 88to 144 mm³ and group mean tumor volumes of 119-121 mm3. Tumors weremeasured in two dimensions using calipers, and volume was calculatedusing the formula:

Tumour Volume (mm³)=0.5 (w²×l)

where w=width and l=length, in mm, of the tumour. Tumour weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumourvolume

Treatment 1

Treatment began on Day 1 in groups of mice (n=10) with establishedsubcutaneous NCI-N87 tumors (88-144 mm³). Trastuzumab-23 wasadministered intravenously once on Day 1 (qd×1) at two dosages (0.3 and1 mg/kg). A vehicle-treated group served as the control group forefficacy analysis. Tumors were measured twice per week until the studywas ended on Day 81. Each mouse was euthanized when its tumor reachedthe endpoint volume of 800 mm³ or on the final day, whichever camefirst. The time to endpoint (TTE) was calculated for each mouse.

Treatment outcome was determined from percent tumor growth delay (%TGD), defined as the percent increase in median TTE for treated versuscontrol mice, with differences between groups deemed statisticallysignificant at P 5 0.05 using log rank survival analysis. Mice weremonitored for complete regression (CR) and partial regression (PR)responses.

Treatment tolerability was assessed by body weight measurements andfrequent observation for signs of treatment-related side effects.Treatment tolerability was assessed by body weight measurements andfrequent observation for signs of treatment-related side effects. Allregimens were acceptably tolerated.

The median TTE for vehicle-treated controls was 53.4 days, establishinga maximum possible TGD of 27.6 days (52%) for the 81-day study.Trastuzumab-23 tested at 1 mg/kg produced survival benefit that wasstatistically significantly different from vehicle-treated controls(P<0.001) and resulted in the maximum possible TGD. At 0.3 mg/kg, themedian TTE was 80.5 days, which corresponds to TGD of 27.1 days (51%).

Trastuzumab-23, at 1 mg/kg produced one partial regressions (PR). Theresults are illustrated in FIG. 3.

Treatment 2

Treatment began on Day 1 in groups of mice (n=10) with establishedsubcutaneous NCI-N87 tumors (75-126 mm³). Trastuzumab-40 wasadministered intravenously once on Day 1 (qd×1) at two dosages (0.3 and1 mg/kg). A vehicle-treated group served as the control group forefficacy analysis. Tumors were measured twice per week until the studywas ended on Day 83. Each mouse was euthanized when its tumor reachedthe endpoint volume of 800 mm³ or on the final day, whichever camefirst. The time to endpoint (TTE) was calculated for each mouse.

Treatment outcome was determined from percent tumor growth delay (%TGD), defined as the percent increase in median TTE for treated versuscontrol mice, with differences between groups deemed statisticallysignificant at P≤0.05 using log rank survival analysis. Mice weremonitored for complete regression (CR) and partial regression (PR)responses. Treatment tolerability was assessed by body weightmeasurements and frequent observation for signs of treatment-relatedside effects. Treatment tolerability was assessed by body weightmeasurements and frequent observation for signs of treatment-relatedside effects. All regimens were acceptably tolerated.

The median TTE for vehicle-treated controls was 44.9 days, establishinga maximum possible TGD of 38.1 days (85%) for the 83-day study.Trastuzumab-40 (0.3 mg/kg) had a median TTE of 54.2 days correspondingto a TGD of 9.3 days (21%). Trastuzumab-40 (1 mg/kg) had a median TTE of61.6 days corresponding to a TGD of 16.7 days (37%).

The results are illustrated in FIG. 4.

Example 8—Toxicity Studies/Therapeutic Index

Rat Study:

A single dose toxicity study was used to determine the maximum tolerateddose (MTD) and safety profile of Trastuzumab-23. Male Sprague Dawleyrats (Harlan, Inc) were dosed once by slow bolus intravenous injectionvia the tail vein with vehicle control (25 mM Histidine-HCl, 7% sucrose,0.02% Polysorbate 80, pH 6.0) or test material (Trastuzumab-23).Parameters evaluated during the study included mortality, physicalexaminations, cageside observations, body weights, body weight changes,clinical pathology (clinical chemistry, hematology, and coagulation),and gross pathology findings. All animals were terminated on Study Day(SD) 29.

Male Rats Dose Dose Main Study Group Treatment Route (mg/kg) Frequency N1 Control IV 0 Single 5 7 Trastuzumab-23 IV 3 Single 5 8 Trastuzumab-23IV 4 Single 5 9 Trastuzumab-23 IV 5 Single 5 10 Trastuzumab-23 IV 6Single 5 13 Trastuzumab-23 IV 7 Single 5 Control = 25 mM Histidine-HCl,7% sucrose, 0.02% Polysorbate 80, pH 6.0

Tolerability was determined based on toxicity end points, including bodyweight loss (>10%) and bone marrow suppression. Based on minimal adversefindings at the high dose, the maximum tolerated dose (MTD) in the ratafter a single dose of Trastuzumab-23 was determined to be >7 mg/kg,which was the highest dose level evaluated.

Therapeutic Index

The Therapeutic Index can be calculated by dividing the maximumtolerated single dose (MTD) of non-targeted ADC in rat, by the minimaleffective single dose (MED) of the a targeted ADC. The MED is the singledose necessary to achieve tumour stasis in an in vivo model at 28 days(for NCI-N87 xenograft).

Thus for conjugates of compound 23, the therapeutic index is the MTD ofgreater than 7 mg/kg divided by the MED which is less than 1 mg/kg (seeFIG. 3 at 28 days), giving a Therapeutic Index of greater than 7.

Cynomolgus Macaque Study:

A Single-Dose Toxicity study was performed in male Cynomolgus macaquesmonkeys (Macaca fascicularis) of Cambodian origin following a singleintravenous (IV) bolus injection of ADC-SG3400. The study was conductedin 2 phases. In phase 1, animals (n=1) were treated at dose levels of 1,3, or 6 mg/kg to determine the optimal dose level to explore in phase 2.Animals were dosed by slow bolus intravenous injection via the saphenousvein with vehicle control (25 mM Histidine, 200 mM Sucrose, pH 6.0) ortest material (Trastuzumab-23). Based on observations of significantbody weight loss at 6 mg/kg, a dose level of 4.5 mg/kg was chosen forphase 2 of the study. In phase 2, animals (n=3) were administered asingle dose of 4.5 mg/kg Trastuzumab-23 on Day 1 and necropsied on Days71 or 72.

Male Cyno Dose Dose Main Study Group Treatment Route (mg/kg) Frequency N1 Trastuzumab-23 IV 1 Single 1 2 Trastuzumab-23 IV 3 Single 1 3Trastuzumab-23 IV 6 Single 1 4 Trastuzumab-23 IV 4.5 Single 3

Tolerability was determined based on toxicity end points, including bodyweight loss (>10%) and bone marrow suppression. There was no unscheduledmortality in any animal administered Trastuzumab-23. The major findingswere body weight loss and bone marrow suppression at the highest testeddose of 6 mg/kg. Based on these data, and minimal signs of toxicity atnext lowest dose, the MTD of Trastuzumab-23 in cynos was 4.5 mg/kg.

All documents and other references mentioned above are hereinincorporated by reference.

1. A conjugate of formula I:L-(D^(L))_(p)  (I) wherein L is a Ligand unit, D is a Drug Linker unitof formula II:

wherein either: (a) R¹⁰ and R¹¹ form a nitrogen-carbon double bondbetween the nitrogen and carbon atoms to which they are bound; or (b)R¹⁰ is OH, and R¹¹ is:

p is an integer of from 1 to
 20. 2. A conjugate according to claim 1,wherein D^(L) is D^(L)-A:


3. A conjugate according to claim 1, wherein D^(L) is D^(L)-B:


4. A conjugate according to according to claim 1, wherein the LigandUnit is an antibody or an active fragment thereof.
 5. The conjugateaccording to claim 4, wherein the antibody or antibody fragment is anantibody or antibody fragment for a tumour-associated antigen.
 6. Theconjugate according to claim 4 wherein the antibody or antibody fragmentis an antibody which binds to one or more tumor-associated antigens orcell-surface receptors selected from (1)-(88): (1) BMPR1B; (2) E16; (3)STEAP1; (4) 0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8) PSCA hIg; (9)ETBR; (10) MSG783; (11) STEAP2; (12) TrpM4; (13) CRIPTO; (14) CD21; (15)CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20R-alpha; (21)Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R;(27) CD22; (28) CD79a; (29) CXCR5; (30) HLA-DOB; (31) P2X5; (32) CD72;(33) LY64; (34) FcRH1; (35) IRTA2; (36) TENB2; (37) PSMA—FOLH1; (38)SST; (38.1) SSTR2; (38.2) SSTR5; (38.3) SSTR1; (38.4) SSTR3; (38.5)SSTR4; (39) ITGAV; (40) ITGB6; (41) CEACAM5; (42) MET; (43) MUC1; (44)CA9; (45) EGFRvIII; (46) CD33; (47) CD19; (48) IL2RA; (49) AXL; (50)CD30—TNFRSF8; (51) BCMA—TNFRSF17; (52) CT Ags—CTA; (53) CD174 (LewisY)—FUT3; (54) CLEC14A; (55) GRP78—HSPA5; (56) CD70; (57) Stem Cellspecific antigens; (58) ASG-5; (59) ENPP3; (60) PRR4; (61) GCC—GUCY2C;(62) Liv-1—SLC39A6; (63) 5T4; (64) CD56—NCMA1; (65) CanAg; (66) FOLR1;(67) GPNMB; (68) TIM-1—HAVCR1; (69) RG-1/Prostate tumor targetMindin—Mindin/RG-1; (70) B7-H4—VTCN1; (71) PTK7; (72) CD37; (73)CD138—SDC1; (74) CD74; (75) Claudins—CLs; (76) EGFR; (77) Her3; (78)RON—MST1R; (79) EPHA2; (80) CD20—MS4A1; (81) Tenascin C—TNC; (82) FAP;(83) DKK-1; (84) CD52; (85) CS1—SLAMF7; (86) Endoglin—ENG; (87) AnnexinA1—ANXA1; (88) V-CAM (CD106)—VCAM1.
 7. The conjugate according to claim4, wherein the antibody or antibody fragment is a cysteine-engineeredantibody.
 8. The conjugate according to claim 1, wherein p is an integerfrom 1 to
 8. 9. The conjugate according to claim 8, wherein p is 1, 2,3, or
 4. 10. A composition comprising a mixture of conjugates accordingto claim 1, wherein the average p in the mixture of conjugate compoundsis about 1 to about
 8. 11. (canceled)
 12. A pharmaceutical compositioncomprising the conjugate of claim 1 and a pharmaceutically acceptablediluent, carrier, or excipient. 13.-15. (canceled)
 16. A method ofmedical treatment comprising administering to a patient thepharmaceutical composition of claim
 12. 17. The method of claim 16,wherein the method of medical treatment is for treating cancer.
 18. Themethod of claim 17, wherein the patient is administered achemotherapeutic agent, in combination with the conjugate. 19.(canceled)
 20. A method of treating a mammal having a proliferativedisease, comprising administering an effective amount of a conjugateaccording to claim
 1. 21. A compound of formula III:

wherein either: (a) R¹⁰ and R¹¹ form a nitrogen-carbon double bondbetween the nitrogen and carbon atoms to which they are bound; or (b)R¹⁰ is OH, and R¹¹ is:


22. A compound according to claim 21 which is A:


23. A compound according to claim 21 which is B:


24. A method of synthesizing a conjugate comprising conjugating acompound according to claim 21 with a cell-binding agent.